A pharmaceutical composition comprising a cell population containing adipose-derived vascular endothelial (precursor) cells

Abstract

To provide a pharmaceutical composition for preventing and / or treating stem cell wasting disease.SOLUTION: A pharmaceutical composition for preventing and / or treating stem cell wasting disease, contains a cell population containing a cultured fat-derived endothelial (progenitor) cell, and a pharmaceutically acceptable medium.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition containing a cell population containing adipose-derived vascular endothelial (progenitor) cells.

Background Art

[0002] Vascular endothelial (progenitor) cells (Endothelial progenitor cell; EPC) have been reported to exist in tissues throughout the human body such as adipose tissue, umbilical vein, and large blood vessels, and are expected to be candidates for cell therapy tools. Since it was unrealistic to use blood vessels in the human body as a raw material for vascular endothelial (progenitor) cells derived from blood vessels, they have not been implemented as cell therapy tools in society until now. On the other hand, adipose-derived vascular endothelial (progenitor) cells (Adipose-derived endothelial progenitor cell; AEPC) can be abundantly obtained from adipose tissue, which can be collected relatively minimally invasively, and thus are regarded as promising candidates for cell therapy tools.

[0003] Conventionally, it has been considered difficult to purify adipose-derived vascular endothelial (progenitor) cells in vitro. Adipose-derived vascular endothelial (progenitor) cells are separated and recovered from adipose tissue as a mixture (stromal vascular fraction; SVF) with other cells such as adipose-derived stem / stromal cells (Adipose-derived stem / stromal cell; ASC), adipose-derived fibroblasts, and adipose-derived pericytes. When attempting to purify the adipose-derived vascular endothelial (progenitor) cells contained in this stromal vascular fraction by general culture techniques, the adipose-derived vascular endothelial (progenitor) cells tend to be displaced by adipose-derived stem cells with much higher proliferative ability.

[0004] The inventors of the present invention have developed for the first time in the world a method for purifying vascular endothelial (precursor) cells, which are abundant in human adipose tissue, in vitro. Patent Document 1 by the inventors of the present invention describes a method for producing a cell population containing vascular endothelial (precursor) cells. The production method described in Patent Document 1 includes: (1) a step of subjecting adipose tissue to enzymatic treatment to obtain a cell population containing at least vascular endothelial (precursor) cells and adipose stem cells; (2) a step of obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (1); (3) a step of culturing the cell population containing the CD31-positive cells obtained in the step (2) for 1 hour to 7.0 days; and (4) a step of obtaining a cell population containing CD31-positive cells by selecting CD31-positive cells from the cell population obtained in the step (3). It is a method for producing a cell population containing vascular endothelial (precursor) cells. Further, Patent Document 1 describes a cell population containing vascular endothelial (precursor) cells and adipose stem cells, wherein the proportion of CD45-negative and CD31-positive vascular endothelial (precursor) cells in the cell population is 92% or more, and the proportion of adipose stem cells in the cell population is 8% or less, and a pharmaceutical composition containing the cell population.

[0005] Patent Document 2 describes a method for preparing endothelial cells from adipose tissue. The method described in Patent Document 2 includes: a step of washing adipose tissue obtained from a liposuction patient; a step of recovering cells from the washed adipose tissue; a step of enzymatically treating the cells with a purified collagenase preparation that lacks pepsin, trypsin, and thermolysin; a step of sorting the treated cells by contacting them with magnetic beads containing a first antibody specific to an antigen selected from a first group consisting of CD31, CD34, CD144, and CD146, or a first antibody specific to an antigen selected from a second group consisting of CD14, CD45, and F19; and a step of recovering the cells bound to the magnetic beads if the antibody is specific to an antigen in the first group, or recovering the cells not bound to the magnetic beads if the antibody is specific to the second group.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2019-088279 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-528841 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] An object of the present invention is to provide a new medical use using adipose-derived vascular endothelial (precursor) cells (AEPC). Specifically, an object of the present invention is to provide a pharmaceutical composition for preventing and / or treating stem cell depletion diseases, which contains cultured adipose-derived vascular endothelial (precursor) cells. [Means for Solving the Problems]

[0008] As a result of intensive studies to solve the above problems, the present inventors surprisingly found that cultured adipose-derived vascular endothelial (precursor) cells (AEPC) have both stem cell properties and lumen-forming ability, and show a remarkable healing effect in a test using a stem cell depletion disease model. Furthermore, the present inventors found that a more remarkable healing effect is shown by administering cultured adipose-derived vascular endothelial (precursor) cells together with cultured adipose-derived stem cells to a stem cell depletion disease model. The present invention has been completed based on these research results.

[0009] That is, according to the present specification, the following inventions are provided. [1] A pharmaceutical composition for preventing and / or treating a stem cell depletion disease, which contains a cell population containing cultured adipose-derived vascular endothelial (precursor) cells and a pharmaceutically acceptable medium. [2] The pharmaceutical composition according to [1], wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells that have been adherently cultured at least twice. [3] The pharmaceutical composition according to [1] or [2], wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (6). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) A step of obtaining a cell population containing adherent cells by subjecting the cell population obtained in the step (1) to adherent culture for 1 hour or more and 7 days or less; (3) A step of obtaining a cell population containing cells positive for CD31 by selecting cells positive for CD31 from the cell population obtained in the step (2); (4) A step of obtaining a cell population containing adherent cells by subjecting the cell population obtained in the step (3) to adherent culture for 1 hour or more and 7 days or less; (5) A step of obtaining a cell population containing cells positive for CD31 by selecting cells positive for CD31 from the cell population obtained in the step (4); and (6) A step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by subjecting the cell population obtained in the step (5) to adherent culture: [4] The pharmaceutical composition according to [1] or [2], wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (5). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) A step of obtaining a cell population containing cells positive for CD31 by selecting cells positive for CD31 from the cell population obtained in the step (1); (3) A step of obtaining a cell population containing adherent cells by subjecting the cell population obtained in the step (2) to adherent culture for 1 hour or more and 7 days or less; (4) A step of obtaining a cell population containing cells positive for CD31 by selecting cells positive for CD31 from the cell population obtained in the step (3); (5) The step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherent culturing of the cell population obtained in the step (4) above: [5] The pharmaceutical composition according to [1] to [4], wherein the cell population contains 50% or more of the adipose-derived vascular endothelial (precursor) cells. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the cell population further contains cultured adipose-derived stem cells. [7] The pharmaceutical composition according to [6], wherein the cell population contains 12.5% or more and 50% or less of the adipose-derived stem cells. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the stem cell-depleting disease is at least one selected from the group consisting of type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer, radiation ulcer, liver cirrhosis, and vascular lesion. [9] The single dose of adipose-derived vascular endothelial (precursor) cells administered to a human is 10 9 cells / kg body weight or less. The pharmaceutical composition according to any one of [1] to [8].

[10] The pharmaceutical composition according to any one of [1] to [9], wherein the pharmaceutical composition is a preparation for injection.

[11] Use of a cell population containing cultured adipose-derived vascular endothelial (precursor) cells for the manufacture of a pharmaceutical composition for preventing and / or treating stem cell-depleting diseases.

[12] The use according to

[11] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells adherently cultured at least twice.

[13] The use according to

[11] or

[12] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (6). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) A step of obtaining a cell population containing adherent cells by adherent culturing the cell population obtained in the step (1) above for 1 hour or more and 7 days or less; (3) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (2); (4) A step of obtaining a cell population containing adherent cells by adherent culturing the cell population obtained in the step (3) for 1 hour or more and 7 days or less; (5) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (4); and (6) A step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherent culturing the cell population obtained in the step (5):

[14] The use according to

[11] or

[12] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (5). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by enzymatically treating adipose tissue; (2) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (1); (3) A step of obtaining a cell population containing adherent cells by adherent culturing the cell population obtained in the step (2) for 1 hour or more and 7 days or less; (4) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (3); (5) A step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherent culturing the cell population obtained in the step (4):

[15] The use according to any one of

[11] to

[14] , wherein the cell population contains 50% or more of the adipose-derived vascular endothelial (precursor) cells.

[16] The use according to any one of

[11] to

[15] , wherein the cell population further contains cultured adipose-derived stem cells.

[17] The use according to

[16] , wherein the cell population contains 12.5% or more and 50% or less of the adipose-derived stem cells.

[18] The use according to any one of

[11] to

[17] , wherein the stem cell-consuming disease is at least one selected from the group consisting of type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer, radiation ulcer, liver cirrhosis, and vascular lesion.

[19] The single dose of adipose-derived vascular endothelial (precursor) cells administered to a human is 10 9 cells / kg body weight or less, and the use according to any one of

[11] to

[18] .

[20] The use according to any one of

[11] to

[19] , wherein the pharmaceutical composition is a preparation for injection.

[21] A cell population containing cultured adipose-derived vascular endothelial (precursor) cells for use in preventing and / or treating stem cell-consuming diseases.

[22] The cell population according to

[21] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial precursor cells that have been adherently cultured at least twice.

[23] The cell population according to

[21] or

[22] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (6). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) A step of obtaining a cell population containing adherent cells by adherently culturing the cell population obtained in the step (1) for 1 hour or more and 7 days or less; (3) A step of obtaining a cell population containing cells positive for CD31 by sorting cells positive for CD31 from the cell population obtained in the step (2); (4) A step of obtaining a cell population containing adherent cells by adherently culturing the cell population obtained in the step (3) for 1 hour or more and 7 days or less; (5) A step of obtaining a cell population containing cells positive for CD31 by sorting cells positive for CD31 from the cell population obtained in the step (4); and (6) The step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherent culture of the cell population obtained in the step (5):

[24] The cell population according to

[21] or

[22] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (5). (1) The step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) The step of obtaining a cell population containing cells that are positive for CD31 by sorting cells that are positive for CD31 from the cell population obtained in the step (1); (3) The step of obtaining a cell population containing adherent cells by subjecting the cell population obtained in the step (2) to adherent culture for 1 hour or more and 7 days or less; (4) The step of obtaining a cell population containing cells that are positive for CD31 by sorting cells that are positive for CD31 from the cell population obtained in the step (3); (5) The step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by subjecting the cell population obtained in the step (4) to adherent culture:

[25] The cell population according to any one of

[21] to

[24] , wherein the cell population contains 50% or more of the adipose-derived vascular endothelial (precursor) cells.

[26] The cell population according to any one of

[21] to

[25] , wherein the cell population further contains cultured adipose-derived stem cells.

[27] The cell population according to

[26] , wherein the cell population contains 12.5% or more and 50% or less of the adipose-derived stem cells.

[28] The cell population according to any one of

[21] to

[27] , wherein the stem cell depletion disease is at least one selected from the group consisting of type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer, radiation ulcer, liver cirrhosis, and vascular lesion.

[29] The single dose of adipose-derived vascular endothelial (precursor) cells administered to a human is 10 9 cells / kg body weight or less, and the cell population according to any one of

[21] to

[28] .

[30] The cell population according to any one of

[21] to

[29] , wherein the pharmaceutical composition is a preparation for injection.

[31] A method for preventing and / or treating a stem cell depletion disease, comprising administering to a patient in need of prevention and / or treatment of a stem cell depletion disease a cell population containing cultured adipose-derived vascular endothelial (precursor) cells.

[32] The method according to

[31] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells that have been adherently cultured at least twice.

[33] The method according to

[31] or

[32] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (6). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) A step of obtaining a cell population containing adherent cells by adherently culturing the cell population obtained in the step (1) for 1 hour or more and 7 days or less; (3) A step of obtaining a cell population containing cells positive for CD31 by selecting cells positive for CD31 from the cell population obtained in the step (2); (4) A step of obtaining a cell population containing adherent cells by adherently culturing the cell population obtained in the step (3) for 1 hour or more and 7 days or less; (5) A step of obtaining a cell population containing cells positive for CD31 by selecting cells positive for CD31 from the cell population obtained in the step (4); and (6) A step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherently culturing the cell population obtained in the step (5):

[34] The method according to

[31] or

[32] , wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial (precursor) cells produced by a production method including the following steps (1) to (5). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (1); (3) A step of obtaining a cell population containing adherent cells by culturing the cell population obtained in the step (2) by adherent culture for 1 hour or more and 7 days or less; (4) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (3); (5) A step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by culturing the cell population obtained in the step (4) by adherent culture:

[35] The method according to any one of

[31] to

[34] , wherein the cell population contains 50% or more of the adipose-derived vascular endothelial (precursor) cells.

[36] The method according to any one of

[31] to

[35] , wherein the cell population further contains cultured adipose-derived stem cells.

[37] The method according to

[36] , wherein the cell population contains 12.5% or more and 50% or less of the adipose-derived stem cells.

[38] The method according to any one of

[31] to

[37] , wherein the stem cell-depleting disease is at least one selected from the group consisting of type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer, radiation ulcer, liver cirrhosis, and vascular lesion.

[39] The single dose of adipose-derived vascular endothelial (precursor) cells administered to a human is 10 9 cells / kg body weight or less, and the method according to any one of

[31] to

[38] .

[40] The method according to any one of

[31] to

[39] , wherein the pharmaceutical composition is a preparation for injection. [Advantages of the Invention]

[0010] According to the present invention, a pharmaceutical composition for preventing and / or treating stem cell-depleting diseases can be provided. [Brief Description of the Drawings]

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments of the present invention will be specifically described. However, the following description is for facilitating the understanding of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which those skilled in the art appropriately substitute the configurations of the following embodiments are also included in the scope of the present invention.

[0013] 1. Explanation of Terms The following terms frequently used in this specification are defined, and their configurations are specifically explained. Unless otherwise specified, the following definitions described in this section are common to other aspects of the present invention. In this specification, "adipose tissue" is a type of connective tissue that constitutes the living body of an organism and mainly exists subcutaneously. Adipose tissue mainly contains mature adipocytes, stores energy, protects the body against physical impacts and temperature changes from the outside world, and has functions such as secreting hormones, cytokines, etc. In this specification, "adipose tissue" may be described as "fat".

[0014] In this specification, "vascular endothelial (progenitor) cells (Endothelial progenitor cell; EPC)" are cells that form the inner surface of blood vessels and are in contact with the lumen through which blood circulates. The "vascular endothelial (progenitor) cells (Endothelial progenitor cell; EPC)" in this specification means a concept that includes vascular endothelial cells and vascular endothelial progenitor cells. In this specification, "adipose-derived endothelial progenitor cells (Adipose-derived endothelial progenitor cell; AEPC)" means vascular endothelial (progenitor) cells derived from adipose tissue. That is, the "adipose-derived endothelial progenitor cells" in this specification means a concept that includes adipose-derived vascular endothelial cells and adipose-derived vascular endothelial progenitor cells.

[0015] In this specification, "adipose-derived vascular endothelial cells" refers to cells that satisfy the following definitions (1) to (5). Definition of Adipose-Derived Vascular Endothelial Cells (1) Derived from adipose tissue. (2) Show adhesiveness to plastic under culture conditions in a standard medium. (3) Are positive for CD31, CD146, and CD105 in flow cytometry. (4) Are negative for CD45 in flow cytometry. It has the ability (lumen formation ability) to form a tubular network structure in a network formation assay. Adipose-derived vascular endothelial cells may be positive for isolectin and / or von Willebrand factor (VWF) in a cell immunostaining test.

[0016] As used herein, "adipose-derived vascular endothelial progenitor cells" refers to cells that satisfy the following definitions (1) to (6). Definition of adipose-derived vascular endothelial progenitor cells (1) It is derived from adipose tissue. (2) It shows adhesiveness to plastic under culture conditions in a standard medium. (3) It is positive for CD31, CD146, and CD105 in flow cytometry. (4) It is negative for CD45 in flow cytometry. (5) It has the ability (lumen formation ability) to form a tubular network structure in a network formation assay. (6) It has the ability (colony formation ability) to form colonies in a colony formation assay. Adipose-derived vascular endothelial progenitor cells may be positive for isolectin and / or von Willebrand factor (VWF) in a cell immunostaining test.

[0017] As used herein, "stem cell" refers to a cell having the ability to differentiate into various cells and the ability of self-renewal. As used herein, "adult stem cell" refers to a stem cell that exists in each tissue of an adult, whose terminal differentiation is incomplete, and which has a certain degree of pluripotency, and is also called a somatic stem cell or a tissue stem cell. As used herein, "adipose-derived stem / stromal cell (ASC)" refers to somatic stem cells derived from adipose tissue and refers to cells that satisfy the following definitions (1) to (4). Definition of Adipose-Derived Stem Cells (1) Derived from adipose tissue. (2) Show adhesiveness to plastic under culture conditions in a standard medium. (3) Show positive for CD90, CD73 and CD105 in flow cytometry. (4) Show negative for CD31 and CD45 in flow cytometry. Adipose-derived stem cells may have the ability to differentiate into adipocytes, osteoblasts, chondroblasts, myofibroblasts, osteocytes, muscle cells or nerve cells, etc.

[0018] As used herein, "adhesive cells" refers to cells that show adhesiveness to plastic under culture conditions in a standard medium. Examples of "adhesive cells" in this specification include adipose-derived vascular endothelial (precursor) cells, adipose-derived stem cells, etc. As used herein, "cell population" refers to a population composed of a plurality of cells including at least the adipose-derived vascular endothelial (precursor) cells. The cell population may be composed only of adipose-derived vascular endothelial (precursor) cells, or may contain cells other than adipose-derived vascular endothelial (precursor) cells. Cells other than adipose-derived vascular endothelial (precursor) cells are preferably cultured adipose-derived stem cells. The adipose-derived vascular endothelial (precursor) cells and / or cells other than adipose-derived vascular endothelial (precursor) cells constituting the cell population can exist separately from each other in a liquid such as a culture solution in the cell population. The liquid containing the cell population in this case is often referred to as "cell suspension" in this specification.

[0019] "CD31" as used herein means cluster of differentiation 31, a kind of surface antigen, and is a protein also known as PECAM-1 (Platelet endothelial adhesion molecule-1). As used herein, "CD146" refers to cluster of differentiation 146, a type of surface antigen, and is a protein also known as MCAM (Melanoma cell adhesion molecule). As used herein, "CD105" refers to cluster of differentiation 105, a type of surface antigen, and is a protein also known as Endoglin. As used herein, "CD45" refers to cluster of differentiation 45, a type of surface antigen, and is a protein also known as PTPRC (Protein tyrosine phosphatase, receptor type, C), or LCA (Leukocyte common antigen).

[0020] As used herein, "CD90" refers to cluster of differentiation 90, a type of surface antigen, and is a protein also known as Thy-1. As used herein, "CD73" refers to cluster of differentiation 73, a type of surface antigen, and is a protein also known as 5-Nucleotidase, or Ecto-5’-nucleotidase. As used herein, "CD34" refers to cluster of differentiation 34, a type of surface antigen, and is a protein also known as Hematopoietic progenitor cell antigen CD34.

[0021] As used herein, "isolectin" refers to a type of protein that exhibits binding activity to sugar chains. As used herein, "von Willebrand factor (VWF)" is a type of plasma glycoprotein.

[0022] As used herein, the "ratio of cells presenting a positive surface antigen" refers to the ratio of cells that are positive for the surface antigen analyzed by flow cytometry. In this specification, the "ratio of cells presenting a positive surface antigen" may be referred to as the "positive rate". As used herein, the "ratio of cells presenting a negative surface antigen" refers to the ratio of cells that are negative for the surface antigen analyzed by flow cytometry. In this specification, the "ratio of cells presenting a negative surface antigen" may be referred to as the "negative rate". Note that the negative rate can be calculated by the formula "negative rate (%) = 100 - positive rate".

[0023] The measurement of the above positive rate can be performed by flow cytometry using a fluorescently labeled antibody. When cells emitting stronger fluorescence are detected as compared with a negative control (isotype control), the cells are determined to be "positive" for the surface antigen. Any antibody can be used as the fluorescently labeled antibody, and examples include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

[0024] As used herein, the "cell viability" refers to the ratio of viable cells. The cell viability can be measured, for example, by Acridine orange / Propidium iodide fluorescence staining, trypan blue staining, MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium Bromide) assay, etc., but is not limited thereto.

[0025] As used herein, "adherent culture" refers to one of the cell culture methods, which means growing cells in a plastic culture vessel in an adherent state in a medium. As used herein, "proliferation" means that cells perform cell division by culture and the number of cells increases. As used herein, "seeding" means sowing cells in a plastic culture vessel for culturing cells.

[0026] As used herein, the "medium" refers to a liquid, semi-solid or solid substance prepared for culturing cells, and in principle contains components essential for cell growth and / or maintenance to a level above the minimum requirement. Unless otherwise specified, the medium used herein refers to a liquid medium for animal cells used for culturing animal-derived cells. As used herein, the "patient or subject" is typically a human, but may also be a non-human animal. Examples of non-human animals include mammals such as dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese macaques), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), etc., and birds such as chickens, quails, etc., but are not limited thereto.

[0027] As used herein, the "pharmaceutically acceptable vehicle" refers to a liquid that can be administered to a patient or subject. Examples of the "stem cell depletion disease" as used herein include, but are not limited to, type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer, radiation ulcer, cirrhosis, vascular lesion, etc.

[0028] As used herein, "treatment" refers to curing the disease of a patient or subject. Examples of "treatment" as used herein include, but are not limited to, significantly improving at least one of the life prognosis, functional prognosis, survival rate, weight loss, fever, anorexia, nutritional disorder, vomiting, fatigue, inflammation, eczema, rash, ulcer, erosion, blister, pain, numbness, spasm, paralysis, ache, itching, dryness, anemia, bleeding, liver function decline, or deterioration of blood test items of a patient or subject.

[0029] As used herein, "prevention" refers to preventing the onset of a disease in a patient or subject. Examples of "prevention" in this specification include, but are not limited to, significantly suppressing in advance at least one of the onset of the patient's or subject's life prognosis, functional prognosis, survival rate, weight loss, fever, loss of appetite, nutritional disorder, vomiting, fatigue, inflammation, eczema, rash, ulcer, erosion, blister, pain, numbness, spasm, paralysis, ache, itching, dryness, anemia, bleeding, deterioration of liver function, or deterioration of blood test items.

[0030] 2. Pharmaceutical Composition 2-1. Overview The pharmaceutical composition for preventing and / or treating stem cell depletion diseases provided by the present invention is characterized by comprising a cell population containing cultured adipose-derived vascular endothelial (precursor) cells and a pharmaceutically acceptable medium. That is, according to the present invention, there is provided a pharmaceutical composition for preventing and / or treating stem cell depletion diseases, which comprises a cell population containing cultured adipose-derived vascular endothelial (precursor) cells and a pharmaceutically acceptable medium. The pharmaceutical composition of the present invention can be used as a therapeutic agent for stem cell depletion diseases. By administering the pharmaceutical composition of the present invention to a treatment site in an amount capable of measuring the effect, the above-mentioned stem cell depletion diseases can be treated.

[0031] According to the present invention, there is provided the use of a cell population containing cultured adipose-derived vascular endothelial (precursor) cells for the manufacture of a pharmaceutical composition for preventing and / or treating stem cell depletion diseases. According to the present invention, there is provided the use of a cell population containing cultured adipose-derived vascular endothelial (precursor) cells for the manufacture of a therapeutic agent for stem cell depletion diseases. According to the present invention, there is provided a cell population containing cultured adipose-derived vascular endothelial (precursor) cells for use in a pharmaceutical composition for preventing and / or treating stem cell depletion diseases.

[0032] According to the present invention, there is provided a cell population containing cultured adipose-derived vascular endothelial (precursor) cells for administration to a patient or subject for use in preventing and / or treating stem cell depletion diseases. According to the present invention, there is provided a method for administering to a patient or subject suffering from a stem cell depletion disease a therapeutically effective amount of a cell population containing cultured adipose-derived vascular endothelial (precursor) cells, which comprises the step of administering the cell population containing cultured adipose-derived vascular endothelial (precursor) cells to the patient or subject, and a method for treating a stem cell depletion disease in a patient or subject.

[0033] 2-2. Cells The pharmaceutical composition of the present invention contains cultured adipose-derived vascular endothelial (precursor) cells. The cultured adipose-derived vascular endothelial (precursor) cells have both stem cell properties and lumen-forming ability, and have a remarkable healing effect on stem cell depletion diseases, so they can be used for the prevention and / or treatment of stem cell depletion diseases.

[0034] The adipose-derived vascular endothelial (precursor) cells as used herein mean a concept encompassing adipose-derived vascular endothelial cells and adipose-derived vascular endothelial precursor cells. The adipose-derived vascular endothelial cells as used herein are (1) derived from adipose tissue, (2) adhesive to plastic under culture conditions in a standard medium, (3) positive for CD31, CD146 and CD105 in flow cytometry, (4) negative for CD45 in flow cytometry, and (5) have the ability to form a tubular network structure (lumen-forming ability) in a network formation assay. Also, the adipose-derived vascular endothelial precursor cells as used herein are (1) derived from adipose tissue, (2) adhesive to plastic under culture conditions in a standard medium, (3) positive for CD31, CD146 and CD105 in flow cytometry, (4) negative for CD45 in flow cytometry, (5) have the ability to form a tubular network structure (lumen-forming ability) in a network formation assay, and (6) have the ability to form colonies (colony-forming ability) in a colony formation assay.

[0035] The adipose-derived vascular endothelial (precursor) cells in this specification may exhibit positive results for isolectin and / or von Willebrand factor (VWF) in a cell immunostaining test. The adipose-derived vascular endothelial (precursor) cells in this specification may exhibit positive results for CD34 and / or CD90, or may exhibit negative results for CD34 and / or CD90. The adipose-derived vascular endothelial (precursor) cells may be passaged adipose-derived vascular endothelial (precursor) cells. The adipose-derived vascular endothelial (precursor) cells may be autologous, allogeneic or xenogeneic cells, but are preferably autologous cells. The adipose-derived vascular endothelial (precursor) cells are preferably non-recombinant adipose-derived vascular endothelial (precursor) cells. The adipose-derived vascular endothelial (precursor) cells in this specification may be commercially available cells or cells obtained through assignment, or may be newly prepared cells. The adipose-derived vascular endothelial (precursor) cells in this specification may be isolated adipose-derived vascular endothelial cells and / or isolated adipose-derived vascular endothelial precursor cells.

[0036] The source species of the adipose-derived vascular endothelial (precursor) cells in this specification is typically human, but may also be non-human animals. Examples of non-human animals include mammals such as dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese macaques), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), etc., and birds such as chickens, quails, etc., but are not limited thereto.

[0037] The adipose-derived vascular endothelial (precursor) cells in this specification are preferably adipose-derived vascular endothelial precursor cells that have been adherently cultured at least twice. The lower limit of the number of times of adherent culture of the adipose-derived vascular endothelial (precursor) cells is preferably 2 or more times, more preferably 3 or more times, still more preferably 4 or more times, still more preferably 5 or more times, still more preferably 6 or more times. Also, the upper limit of the number of times of adherent culture of the adipose-derived vascular endothelial (precursor) cells is not particularly limited, but may be, for example, 25 or less times, 20 or less times, 15 or less times, or 10 or less times.

[0038] The adipose-derived vascular endothelial (precursor) cells in this specification are preferably adipose-derived vascular endothelial precursor cells that have been sorted at least once. The lower limit of the number of times of sorting of the adipose-derived vascular endothelial (precursor) cells is preferably 1 or more times, more preferably 2 or more times. Also, the upper limit of the number of times of sorting of the adipose-derived vascular endothelial (precursor) cells is not particularly limited, but may be, for example, 6 or less times, 5 or less times, 4 or less times, or 3 or less times. Examples of the method for sorting cells include Fluorescence activated cell sorting (FACS), Magnetic activated cell sorting (MACS), etc. Among the above, MACS is preferred.

[0039] The pharmaceutical composition of the present invention preferably contains cultured adipose-derived stem cells. By using the cultured adipose-derived vascular endothelial (precursor) cells together with the cultured adipose-derived stem cells, the curative effect on stem cell depletion diseases can be significantly enhanced.

[0040] The adipose-derived stem cells in this specification are preferably cells individually cultured in a culture vessel different from the adipose-derived vascular endothelial (precursor) cells in this specification. When the adipose-derived stem cells and the adipose-derived vascular endothelial (precursor) cells are seeded and cultured in the same culture vessel, it is not preferable because the adipose-derived stem cells may drive out the adipose-derived vascular endothelial (precursor) cells due to the difference in proliferation ability.

[0041] The adipose-derived stem cells in this specification are preferably passage-passaged adipose-derived stem cells. The adipose-derived stem cells may be autologous, allogeneic or xenogeneic cells, but are preferably autologous cells. The adipose-derived stem cells are preferably adipose-derived stem cells that have not been genetically modified. The adipose-derived stem cells in this specification may be commercially available cells or cells obtained by assignment, or newly prepared cells. The adipose-derived stem cells in this specification may be isolated adipose-derived stem cells. The adipose-derived stem cells in this specification may be sorted adipose-derived stem cells.

[0042] The biological species from which the adipose-derived stem cells in this specification are derived is typically human, but may also be other animals. Examples of other animals include mammals such as dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese macaques), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), etc., and birds such as chickens, quails, etc., but are not limited thereto. The biological species from which the adipose-derived stem cells in this specification are derived is preferably the same as the biological species from which the adipose-derived vascular endothelial (precursor) cells in this specification are derived.

[0043] The adipose-derived stem cells in this specification can be adipose-derived stem cells that have been adherently cultured at least once. The lower limit of the number of times of adherent culture of adipose-derived stem cells may be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more. Also, the upper limit of the number of times of adherent culture of adipose-derived stem cells is not particularly limited, but may be, for example, 25 or less, 20 or less, 15 or less, or 10 or less.

[0044] 2-3. Method for manufacturing cells The adipose-derived vascular endothelial (precursor) cells in this specification are preferably adipose-derived vascular endothelial (precursor) cells manufactured by the following manufacturing method A or manufacturing method B.

[0045] 2-3-1. Manufacturing method A The adipose-derived vascular endothelial (precursor) cells in this specification can be adipose-derived vascular endothelial (precursor) cells manufactured by a manufacturing method A including the following steps (1) to (6). (1) A step of obtaining a cell population containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by subjecting adipose tissue to enzymatic treatment; (2) A step of obtaining a cell population containing adherent cells by subjecting the cell population obtained in the step (1) to adherent culture for 1 hour or more and 7 days or less; (3) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (2); (4) A step of obtaining a cell population containing adherent cells by adherent culturing the cell population obtained in the step (3) for 1 hour or more and 7 days or less; (5) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (4); and (6) A step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherent culturing the cell population obtained in the step (5):

[0046] By the above production method, adipose-derived vascular endothelial (precursor) cells useful for the prevention and / or treatment of stem cell depletion diseases can be obtained with high efficiency. In addition, the adipose-derived vascular endothelial (precursor) cells produced by the above production method have a remarkable healing effect on stem cell depletion diseases and can be used for the prevention and / or treatment of stem cell depletion diseases.

[0047] 2-3-1-1. Step (1) in Production Method A Step (1) in Production Method A is a step of obtaining a cell population (i.e., stromal vascular fraction (SVF)) containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by enzymatically treating adipose tissue. Adipose tissue can be obtained by surgical resection or aspiration from, for example, humans, non-human mammals, or birds. Examples of non-human mammals include dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese macaques), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), etc. Examples of birds include chickens, quails, etc. When surgically resected, adipose tissue may be locally anesthetized. Also, aspirated adipose tissue can be obtained by inserting a cannula into subcutaneous adipose tissue in the abdomen, thigh, buttocks, or the whole body. The amount of adipose tissue obtained is, for example, 1 g to 1000 g, preferably 1 g to 500 g, 1 g to 100 g, 2 g to 50 g, or 2 g to 40 g, but is not limited thereto.

[0048] It is preferable to visually confirm that the obtained adipose tissue has no tumorous lesions or contamination. The adipose tissue may be confirmed to be negative for HBV, HCV, HIV, HTLV-1, and TPHA / RPR. The adipose tissue may be confirmed to have less than 128-fold mycoplasma (PA method) and less than 320-fold herpes simplex (CF method). When using aspirated adipose tissue, it is preferable to let the aspirated adipose tissue stand to separate the fat layer and the aqueous layer. Also, the aspirated adipose tissue can be separated by a centrifuge to separate the fat layer and the aqueous layer. After the fat layer and the aqueous layer are separated, only the fat layer can be separated by recovering and removing the aqueous layer. The obtained adipose tissue may be washed with, for example, physiological saline and then subjected to enzyme treatment.

[0049] The adipose tissue before being subjected to enzyme treatment may be warmed at room temperature or in a 37°C water bath for 1 minute to 15 minutes before the enzyme treatment. Enzymatic treatment can be carried out by adding an appropriate amount of enzyme reaction solution to the adipose tissue in the tube, fixing the tube on a thermostatic shaker, and shaking it. The temperature of the enzymatic treatment is not particularly limited as long as the enzyme reaction proceeds, but generally it is 25°C to 50°C, preferably 30 to 45°C, for example, 37°C. The shaking can be reciprocating shaking or swirling shaking. In the case of reciprocating shaking, the reciprocating shaking speed is not particularly limited, but generally it is 10 rpm to 300 rpm, preferably 50 rpm to 200 rpm, for example, 120 rpm. The reaction time is not particularly limited, but generally it is 10 minutes to 3 hours, preferably 15 minutes to 1 hour, for example, 30 minutes.

[0050] As the enzyme, it is preferably to use at least collagenase. Collagenase is a recombinant protein produced by animal tissue cells, inflammatory cells, tumor cells or bacteria such as Clostridium histolyticum, or artificially produced by genetic recombination technology, and refers to an enzyme that decomposes type I, type II, and type III collagen.

[0051] The concentration of collagenase in the enzyme treatment solution is preferably 0.02% to 0.5%, more preferably 0.1% to 0.5%, still more preferably 0.1% to 0.4%, still more preferably 0.1% to 0.3%, and most preferably 0.2%. The enzyme treatment solution used for enzymatic treatment preferably further contains DNaseI in addition to collagenase. When using DNaseI, the concentration of DNaseI in the enzyme treatment solution is preferably 100 to 10000 U / mL, more preferably 200 to 5000 U / mL, still more preferably 500 to 2000 U / mL.

[0052] The enzyme treatment solution used for enzymatic treatment preferably further contains CaCl2. The concentration of CaCl2 in the enzyme treatment solution is preferably 1 mM to 10 mM, more preferably 2 mM to 5 mM, still more preferably 2 mM to 4 mM, for example, 3 mM. The enzyme treatment solution used for enzyme treatment is preferably a buffer solution, more preferably HBSS (Hanks’ balanced salt solution) or DPBS (Dulbecco‘s Phosphate Buffer Saline). A preferred specific example of the composition of the enzyme treatment solution used for enzyme treatment is 0.2% collagenase, HBSS, 3 mM CaCl2, and 1000 U / ml DNaseI.

[0053] 2-3-1-2. Step (2) in Production Method A Step (2) in Production Method A is a step of obtaining a cell population containing adherent cells by adherent culturing the cell population obtained in step (1) (i.e., the stromal vascular fraction (SVF)) for 1 hour or more and 7 days or less. By step (2), while significantly preventing the expulsion of adipose-derived vascular endothelial (precursor) cells due to the proliferation of adipose-derived stem cells, blood cell-derived cells contained in the supernatant can be removed, whereby a cell population containing adipose-derived vascular endothelial (precursor) cells can be obtained.

[0054] The lower limit of the period of adherent culturing in step (2) is preferably 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, or 1 day or more. The upper limit of the period of adherent culturing in step (2) can be 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, 42 hours or less, 36 hours or less, or 30 hours or less. The culturing conditions in step (2) can be set according to the conditions suitable for culturing normal animal cells (preferably vascular endothelial (precursor) cells).

[0055] The medium is not particularly limited as long as it can culture vascular endothelial (progenitor) cells, and examples include EGM-2 medium (Lonza), EGM-2MV medium (Lonza), αMEM, Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified Eagle's medium / Ham's F-12 mixed medium (DMEM / F12), RPMI1640, etc. For these culture solutions, various additives applicable to normal cell culture, such as serum, various vitamins, various antibiotics, various hormones, various growth factors, etc., may usually be added. Particularly preferably, EGM-2 medium (Lonza) or EGM-2MV medium (Lonza) can be used as the medium. The culture is preferably carried out using a plastic culture container such as a flask under the conditions of 37°C and 5% CO2. The medium exchange can be performed, for example, daily or every other day.

[0056] 2-3-1-3. Step (3) in Production Method A Step (3) in Production Method A is a step of obtaining a cell population containing cells that are positive for CD31 by selecting cells that are positive for CD31 from the cell population obtained in step (2). Thereby, the content rate of adipose-derived vascular endothelial (progenitor) cells in the cell population can be dramatically increased. Alternatively, step (3) may be a step of obtaining a cell population containing cells that are negative for CD45 and positive for CD31 by selecting cells that are negative for CD45 and positive for CD31 from the cell population obtained in step (2).

[0057] The method for selecting cells that are positive for CD31, or cells that are negative for CD45 and positive for CD31, is not particularly limited, and a method for selecting cells that express a marker protein or do not express a marker protein using an antibody may be used. Cell sorting can be performed using an antibody that can specifically bind to CD31, and further, if desired, an antibody that can specifically bind to CD45. The antibody is not particularly limited as long as it can specifically bind to the above-mentioned marker protein, and it may be either a polyclonal antibody or a monoclonal antibody. Also, the antibody may be a fragment as long as it can specifically bind to the above-mentioned marker protein. Examples of antibody fragments include Fab fragments, F(ab’)2 fragments, single-chain antibodies (scFv), etc.

[0058] Examples of methods for sorting cells that express a marker protein or cells that do not express a marker protein using an antibody include Fluorescence activated cell sorting (FACS), Magnetic activated cell sorting (MACS), etc. Among these, MACS is preferred.

[0059] Magnetic activated cell sorting (MACS) is a technique for separating target cells using magnetism. Specifically, an antibody against a marker protein is immobilized on magnetic beads, and the target cells can be separated in a cylindrical container (column) or a tube using a strong magnet. As the magnetic bead reagent to be immobilized, general ones can be used. Examples of Magnetic activated cell sorting (MACS) include, but are not limited to, MACS (manufactured by Miltenyi Biotec), IMag (manufactured by BD Japan), etc.

[0060] In step (3), cells that are positive for CD31, or cells that are negative for CD45 and positive for CD31 can be sorted by MACS using magnetic beads labeled with an anti-CD31 antibody and, if desired, magnetic beads labeled with an anti-CD45 antibody. In fluorescence-activated cell sorting (FACS), by using a flow cytometer with cell sorter function, it is possible to collect only specific cells that emit designated fluorescence. Examples of such devices include FACSAriaII (manufactured by BD Japan), JSAN (manufactured by Bay Bioscience), MoFlo XDP (manufactured by Beckman Coulter), and the like.

[0061] 2-3-1-4. Step (4) in Production Method A Step (4) in Production Method A is a step of obtaining a cell population containing adherent cells by adherent culture of the cell population obtained in step (3) for 1 hour or longer and 7 days or shorter. Thereby, while significantly preventing the expulsion of adipose-derived vascular endothelial (precursor) cells, it is possible to selectively proliferate adipose-derived vascular endothelial (precursor) cells. The lower limit of the period of adherent culture in step (4) is preferably 1 hour or longer, 2 hours or longer, 4 hours or longer, 6 hours or longer, 12 hours or longer, 18 hours or longer, or 1 day or longer. The upper limit of the period of adherent culture in step (4) can be 7 days or shorter, 6 days or shorter, 5 days or shorter, 4 days or shorter, 3 days or shorter, 2 days or shorter, 42 hours or shorter, 36 hours or shorter, or 30 hours or shorter. The culture conditions in step (4) can be set according to conditions suitable for culturing normal animal cells (preferably vascular endothelial (precursor) cells).

[0062] The medium is not particularly limited as long as it can culture vascular endothelial (precursor) cells, and examples include EGM-2 medium (Lonza), EGM-2MV medium (Lonza), αMEM, Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified Eagle's medium / Ham's F-12 mixed medium (DMEM / F12), RPMI1640, and the like. For these culture solutions, various additives applicable to normal cell culture, such as serum, various vitamins, various antibiotics, various hormones, various growth factors, etc., may usually be added. Particularly preferably, EGM-2 medium (Lonza) or EGM-2MV medium (Lonza) can be used as the medium. The cultivation is preferably carried out under the conditions of 37°C and 5% CO2 using a plastic culture container such as a flask. The medium exchange may be carried out, for example, daily or every other day.

[0063] 2-3-1-5. Step (5) in Production Method A Step (5) in Production Method A is a step of obtaining a cell population containing cells that are positive for CD31 by selecting cells that are positive for CD31 from the cell population obtained in step (4). Thereby, the content rate of adipose-derived vascular endothelial (precursor) cells in the cell population can be further increased significantly. Alternatively, step (5) may be a step of obtaining a cell population containing cells that are negative for CD45 and positive for CD31 by selecting cells that are negative for CD45 and positive for CD31 from the cell population obtained in step (4).

[0064] The method for selecting cells that are positive for CD31, or cells that are negative for CD45 and positive for CD31 is not particularly limited, and a method for selecting cells that express a marker protein or do not express a marker protein using an antibody may be used. From the viewpoints of cell selection efficiency and / or quality control, the method for selecting cells in step (5) is preferably the same as the method for selecting cells in step (3).

[0065] Cell selection can be carried out using an antibody that can specifically bind to CD31, and further, if desired, an antibody that can specifically bind to CD45. The antibody is not particularly limited as long as it can specifically bind to the above marker protein, and may be either a polyclonal antibody or a monoclonal antibody. Also, the antibody may be a fragment as long as it can specifically bind to the above marker protein. Examples of antibody fragments include Fab fragments, F(ab’)2 fragments, single-chain antibodies (scFv), etc.

[0066] As a method for selecting cells expressing a marker protein or cells not expressing a marker protein using an antibody, for example, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), etc. can be mentioned. Among them, MACS is preferable. Magnetic-activated cell sorting (MACS) is a technique for separating target cells using magnetism. Specifically, an antibody against a marker protein is immobilized on magnetic beads, and the target cells can be separated in a cylindrical container (column) or a tube using a strong magnet. As the magnetic bead reagent to be immobilized, common ones can be used. Examples of magnetic-activated cell sorting (MACS) include, but are not limited to, MACS (manufactured by Miltenyi Biotec), IMag (manufactured by BD Japan), etc.

[0067] In step (5), cells that are positive for CD31, or cells that are negative for CD45 and positive for CD31 can be sorted by MACS using magnetic beads labeled with an anti-CD31 antibody and, if desired, magnetic beads labeled with an anti-CD45 antibody. In FACS, by using a flow cytometer having a cell sorter function, it is possible to collect only specific cells that emit a specified fluorescence. Examples of such devices include FACSAriaII (manufactured by BD Japan), JSAN (manufactured by Bay Bioscience), MoFlo XDP (manufactured by Beckman Coulter), etc.

[0068] 2-3-1-6. Step (6) in Production Method A Step (6) in Production Method A is a step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherent culture of the cell population obtained in step (5). Thereby, adipose-derived vascular endothelial (precursor) cells useful for the prevention and / or treatment of stem cell depletion diseases can be selectively and highly efficiently proliferated. The lower limit of the adhesion culture period in step (6) is not particularly limited, and can be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, or 6 days or more. Also, the upper limit of the adhesion culture period in step (6) is not particularly limited, and can be 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less. The culture conditions in step (6) can be set according to the conditions suitable for culturing normal animal cells (preferably vascular endothelial (progenitor) cells).

[0069] The medium is not particularly limited as long as it can culture vascular endothelial (progenitor) cells, and examples include EGM-2 medium (Lonza), EGM-2MV medium (Lonza), αMEM, Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified Eagle's medium / Ham's F-12 mixed medium (DMEM / F12), RPMI1640, etc. For these culture solutions, various additives applicable to normal cell culture, such as serum, various vitamins, various antibiotics, various hormones, various growth factors, etc., can usually be added. Particularly preferably, EGM-2 medium (Lonza) or EGM-2MV medium (Lonza) can be used as the medium. The culture is preferably carried out under the conditions of 37 °C and 5% CO2 using a plastic culture container such as a flask. The medium exchange can be carried out, for example, daily, every other day, every two days, or every three days.

[0070] 2-3-2. Manufacturing method B The adipose-derived vascular endothelial (progenitor) cells in this specification can be the adipose-derived vascular endothelial (progenitor) cells produced by manufacturing method B including the following steps (1) to (5). (1) A step of obtaining a cell population containing at least vascular endothelial (progenitor) cells and adipose-derived stem cells by enzymatically treating adipose tissue; (2) A step of obtaining a cell population containing CD31-positive cells by sorting CD31-positive cells from the cell population obtained in the step (1); (3) The step of obtaining a cell population containing adherent cells by adherent culture of the cell population obtained in the step (2) above for 1 hour or more and 7 days or less; (4) The step of obtaining a cell population containing cells that are positive for CD31 by selecting cells that are positive for CD31 from the cell population obtained in the step (3); (5) The step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by adherent culture of the cell population obtained in the step (4): By the above production method, adipose-derived vascular endothelial (precursor) cells useful for the prevention and / or treatment of stem cell depletion diseases can be obtained with high efficiency. In addition, the adipose-derived vascular endothelial (precursor) cells produced by the above production method have a remarkable healing effect on stem cell depletion diseases and can be used for the prevention and / or treatment of stem cell depletion diseases.

[0071] 2-3-2-1. Step (1) in Production Method B Step (1) in Production Method B is a step of obtaining a cell population (i.e., stromal vascular fraction (SVF)) containing at least vascular endothelial (precursor) cells and adipose-derived stem cells by enzymatic treatment of adipose tissue. Step (1) in Production Method B can be carried out in the same manner as the above-mentioned "2-3-1-1. Step (1) in Production Method A".

[0072] 2-3-2-2. Step (2) in Production Method B Step (2) in Production Method B is a step of obtaining a cell population containing cells that are positive for CD31 by selecting cells that are positive for CD31 from the cell population obtained in step (1). Thereby, the content rate of adipose-derived vascular endothelial (precursor) cells in the cell population can be increased dramatically. Step (2) in Production Method B can be carried out in the same manner as the above-mentioned "2-3-1-3. Step (3) in Production Method A".

[0073] 2-3-2-3. Step (3) in Production Method B Step (3) in Production Method B is a step of obtaining a cell population containing adherent cells by performing adherent culture on the cell population obtained in step (2) for 1 hour or more and 7 days or less. This can significantly prevent the expulsion of adipose-derived vascular endothelial (precursor) cells while selectively proliferating adipose-derived vascular endothelial (precursor) cells. Step (3) in Production Method B can be carried out in the same manner as step (4) in "2-3-1-4. Production Method A" described above.

[0074] 2-3-2-4. Step (4) in Production Method B Step (4) in Production Method B is a step of obtaining a cell population containing cells that are CD31-positive by sorting the cell population obtained in step (3). This can further dramatically increase the content rate of adipose-derived vascular endothelial (precursor) cells in the cell population. Step (4) in Production Method B can be carried out in the same manner as step (5) in "2-3-1-5. Production Method A" described above.

[0075] 2-3-2-5. Step (5) in Production Method B Step (5) in Production Method B is a step of obtaining cultured adipose-derived vascular endothelial (precursor) cells by performing adherent culture on the cell population obtained in step (4). This can selectively and highly efficiently proliferate adipose-derived vascular endothelial (precursor) cells useful for the prevention and / or treatment of stem cell depletion diseases. Step (5) in Production Method B can be carried out in the same manner as step (6) in "2-3-1-6. Production Method A" described above.

[0076] 2-4. Cell population The pharmaceutical composition of the present invention comprises a cell population containing cultured adipose-derived vascular endothelial (precursor) cells. Preferably, the cell population contains 50% or more of the cultured adipose-derived vascular endothelial (precursor) cells. The ratio of the cultured adipose-derived vascular endothelial (precursor) cells in the cell population is preferably 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more. The cell population herein preferably further comprises cultured adipose-derived stem cells. The cell population can contain 12.5% or more, 15% or more, 17.5% or more, 20% or more, 22.5% or more, or 25% or more of the cultured adipose-derived stem cells. Also, the cell population can contain 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less of the cultured adipose-derived stem cells. By the cell population further comprising the cultured adipose-derived stem cells, the curative effect on stem cell depletion diseases can be significantly enhanced.

[0077] In the cell population herein, the ratio of cells presenting positive for CD31 is preferably 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more. In the cell population herein, the ratio of cells presenting positive for CD146 is preferably 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0078] In the cell population herein, the ratio of cells presenting positive for CD105 is preferably 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. In the cell population described in this specification, the ratio of cells presenting positive for CD45 is preferably less than 5%, 4% or less, 3% or less, 2% or less, 1% or less, or 0%. Also, in the cell population described in this specification, the ratio of cells presenting negative for CD45 is preferably 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0079] According to one aspect of the present invention, in the cell population, the ratio of cells presenting positive for CD90 may be 50% or less, 40% or less, 30% or less, 25% or less, 22.5% or less, 20% or less, 17.5% or less, 15% or less, 12.5% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%. According to one aspect of the present invention, in the cell population, the ratio of cells presenting positive for CD73 may be 50% or less, 40% or less, 30% or less, 25% or less, 22.5% or less, 20% or less, 17.5% or less, 15% or less, 12.5% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%.

[0080] According to one aspect of the present invention, in the cell population, the ratio of cells presenting positive for CD34 may be 50% or less, 40% or less, 30% or less, 25% or less, 22.5% or less, 20% or less, 17.5% or less, 15% or less, 12.5% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%. According to one aspect of the present invention, the cell viability of the cell population described in this specification is preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0081] 2 - 5. Pharmaceutical Composition According to the present invention, there is provided a pharmaceutical composition for preventing and / or treating a stem cell depletion disease, comprising a cell population containing cultured adipose-derived vascular endothelial (precursor) cells and a pharmaceutically acceptable medium. The stem cell depletion disease may be at least one selected from the group consisting of type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer, radiation ulcer, cirrhosis, and vascular lesion. That is, the pharmaceutical composition of the present invention may be a pharmaceutical composition for preventing and / or treating type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer, radiation ulcer, cirrhosis, and / or vascular lesion.

[0082] The pharmaceutically acceptable medium herein is not particularly limited as long as it is a liquid that can be administered to a patient or subject. Pharmaceutically acceptable media include, for example, water for injection, physiological saline, medium, 5% glucose solution, hyaluronic acid solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, Bicanate (registered trademark) infusion, amino acid solution, starting solution (solution No. 1), dehydration replenishing solution (solution No. 2), maintenance infusion (solution No. 3), postoperative recovery solution (solution No. 4), Plasma-Lyte A (registered trademark), etc., but are not limited thereto.

[0083] The pharmaceutical composition of the present invention may be an additive that can be administered to a patient or subject, and may contain an additive that can adjust the storage stability, isotonicity, absorbability, and / or viscosity, etc. of the pharmaceutical composition. Examples of the above additives include, but are not limited to, emulsifiers, dispersants, buffers, preservatives, wetting agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, etc. Examples of the thickener include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, etc. The concentration of the additive can be arbitrarily set as long as it is safe when administered to a patient or subject.

[0084] The pharmaceutical composition of the present invention may contain any component that can be administered to a patient or a subject. Examples of the above components include, but are not limited to, salts, polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, culture medium components, etc.

[0085] The pH of the pharmaceutical composition of the present invention can be a pH near neutrality, for example, pH 5.5 or higher, pH 6.0 or higher, pH 6.5 or higher, or pH 7.0 or higher, and can also be pH 10.5 or lower, pH 9.5 or lower, pH 8.5 or lower, or pH 8.0 or lower, but is not limited thereto.

[0086] The cell concentration of the pharmaceutical composition of the present invention varies depending on the administration form, administration method, purpose of use, and age, weight, symptoms, etc. of the patient or subject, but can be any cell concentration that can be administered to the patient or subject. The lower limit of the cell concentration is not particularly limited, but for example, 1.0×10 5 cells / mL or more, 2.0×10 5 cells / mL or more, 4.0×10 5 cells / mL or more, 6.0×10 5 cells / mL or more, 8.0×10 5 cells / mL or more, 1.0×10 6 cells / mL or more, 2.0×10 6 cells / mL or more, 4.0×10 6 cells / mL or more, 6.0×10 6 cells / mL or more, 8.0×10 6 cells / mL or more, or 1.0×10 7 cells / mL or more. The upper limit of the cell concentration is not particularly limited, but for example, 1.0×10 10 cells / mL or less, 1.0×10 9 cells / mL or less, 8.0×10 8 cells / mL or less, 6.0×10 8 cells / mL or less, 4.0×10 8 cells / mL or less, 2.0×10 8 cells / mL or less, or 1.0×10 8 cells / mL or less.

[0087] The pharmaceutical composition of the present invention is preferably a liquid preparation, more preferably a liquid preparation for injection. As liquid preparations for injection, for example, in International Publication WO2011 / 043136, Japanese Unexamined Patent Application Publication No. 2013-256510, etc., liquid preparations suitable for injection are known. The pharmaceutical composition of the present invention can also be a liquid preparation for injection described in the above documents. Further, the above liquid preparation may be a suspension of cells or a liquid preparation in which cells are dispersed in the liquid preparation. Furthermore, the form of the cells contained in the liquid preparation may be, for example, single cells or cell aggregates.

[0088] The administration method of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include subcutaneous injection, intradermal injection, intramuscular injection, intranodal injection, intravenous injection, intraarterial injection, intravenous drip injection, intraperitoneal injection, intrathoracic injection, direct injection into a local area, application to a local area, etc. According to one aspect of the present invention, a liquid preparation for injection can be filled into a syringe and administered intravenously, intraarterially, intramyocardially, intraarticularly, intrahepatically, intramuscularly, epidurally, subgingivally, intraventricularly, subcutaneously, intradermally, intraperitoneally, intraportally, etc. through an injection needle or a catheter. Regarding the administration method of the pharmaceutical composition, for example, in Japanese Unexamined Patent Application Publication No. 2015-61520, Onken JE, et al. American College of Gastroenterology Conference 2006 Las Vegas, NV, Abstract 121., Garcia-Olmo D, et al. Dis Colon Rectum 2005;48:1416-23., etc., intravenous injection, intravenous drip injection, direct injection into a local area, etc. are known. The pharmaceutical composition of the present invention can also be administered by various methods described in the above documents.

[0089] The dosage of the pharmaceutical composition of the present invention is an amount of adipose-derived vascular endothelial (precursor) cells that can achieve a curative effect on stem cell depletion diseases when administered to a patient or subject. The specific dosage can be appropriately determined according to the dosage form, administration method, purpose of use, and the age, weight, symptoms, etc. of the patient or subject. The lower limit of the single dosage of adipose-derived vascular endothelial (precursor) cells in humans is not particularly limited. For example, it is 1×10 4 cells / kg body weight or more, 5×10 4 cells / kg body weight or more, 1×10 5 cells / kg body weight or more, 5×10 5 cells / kg body weight or more, 1×10 6 cells / kg body weight or more, 2×10 6 cells / kg body weight or more, 3×10 6 cells / kg body weight or more, 4×10 6 cells / kg body weight or more, 5×10 6 cells / kg body weight or more. Also, the upper limit of the single dosage of adipose-derived vascular endothelial (precursor) cells in humans is not particularly limited. For example, it is 1×10 9 cells / kg body weight or less, 5×10 8 cells / kg body weight or less, 1×10 8 cells / kg body weight or less, 9×10 7 cells / kg body weight or less, 8×10 7 cells / kg body weight or less, 7×10 7 cells / kg body weight or less, 6×10 7 cells / kg body weight or less, 5×10 7 cells / kg body weight or less, 4×10 7 cells / kg body weight or less, 3×10 7 cells / kg body weight or less, 2×10 7 cells / kg body weight or less.

[0090] The administration frequency of the pharmaceutical composition of the present invention is a frequency that can achieve a curative effect on stem cell depletion diseases when administered to a patient or subject. The specific administration frequency can be appropriately determined according to the administration form, administration method, purpose of use, and the age, weight, symptoms, etc. of the patient or subject. For example, it can be once every 4 weeks, once every 3 weeks, once every 2 weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week.

[0091] The administration period of the pharmaceutical composition of the present invention is a period that can achieve a curative effect on stem cell depletion diseases when administered to a patient or subject. The specific administration period can be appropriately determined according to the administration form, administration method, purpose of use, and the age, weight, symptoms, etc. of the patient or subject. For example, it can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0092] The timing of administering the pharmaceutical composition of the present invention to a patient or subject is not particularly limited. For example, it can be immediately after the onset, within n days from the onset (n represents an integer of 1 or more), immediately after the diagnosis, within n days from the diagnosis (n represents an integer of 1 or more), and the like. The pharmaceutical composition of the present invention can be stored in a frozen state until immediately before use. When administering the pharmaceutical composition of the present invention to a patient or subject, it can be rapidly thawed at 37°C and then used. In addition, the pharmaceutical composition of the present invention can also be used immediately after being manufactured without frozen storage. The present invention will be specifically described in the following examples, but the present invention is not limited by the examples.

Examples

[0093] <Method> 1. Reagents, equipment used, etc. HBSS without Ca++, without Mg++ (Gibco, #14175-095) Crude collagenase purification, derived from Clostridium histolyticum (Wako Pure Chemical Industries, #032-22364) Conical tubes (500 mL, Corning, #431123; 250 mL, Corning #430776; 50 mL, Falcon, #352070, 15 mL, FALCON #352096) Bench centrifuge (Kubota, main body #S700T, stand #RS-7504M, bucket #053-0100) Thermostatic shaker (Yamato, #100) Electronic scale Pipette silicon rubber for 10 mL (AS ONE, #6-356-04) Cell culture dish (Falcon, #353025, 150 mm dish, growth area 156.36 cm 2 ) Cell strainer φ100 μm, FALCON #352360 Cell strainer φ40 μm, FALCON #352340 DNaseI crude purification (Worthington, #LS002138) Blood cell lysis kit (Miltenyi, #130-094-183) BSA fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, ≧96% (agarose gel electrophoresis) (Sigma, #A8806) EDTA-2Na (Dojindo, #N001) MS column (Miltenyi, #120-000-472) MACS separator (Miltenyi)

[0094] CD45 Microbeads (Miltenyi, #130-045-801) CD31 Microbeads kit (human) (Miltenyi, #130-091-935) Culture sure CaCl2 (Wako Pure Chemical Industries, #037-24031, MW110.98) 0.22 μm φ syringe filter (Millipore, Millex-GV, #2LGV-33RS) Medium for vascular endothelial (precursor) cells (EGM-2, Lonza #CC-3162, EGM-2MV, Lonza #CC-3202) Medium for adipose-derived stem cells (DMEM / F12, Wako Pure Chemical #048-29785) TrypLE express (Gibco, #12604-021) Dimethyl sulfoxide for molecular biology (DMSO) (Wako Pure Chemical, #047-29353) Fetal Bovine Serum (FBS) Penicillin-Streptomycin (Wako Pure Chemical, #168-23191) Cryopreservation unit (Thermo Fisher Scientific, #5100-0001) -80 °C deep freezer, gas-phase liquid nitrogen ultra-low temperature freezer Sonicator (Branson, #M2800J) Disposable pipette (Corning, Costar, 5 mL #4487, 10 mL #4488, 25 mL #4489, 50 mL #4490) Matrigel basement membrane matrix phenol red-free (Corning, #356237) 96-well plate, SpectraPlate-96 TC (PerkinElmer, #6005650) 4% Paraformaldehyde·phosphate buffer (Wako Pure Chemical, #163-20145) Glycine (Wako Pure Chemical, #077-00735) Triton-X100 (Wako Pure Chemical, #591-12191) Human CD31 antibody (R&D, #BBA7) Normal mouse IgG antibody (Santa cruz, #sc-2025) Alexa-488 Goat anti-mouse IgG1 antibody (Invitrogen, #A21121) DAPI (Dojindo, #340-07971) 4-well chamber slide (Iwaki, #5722-004) VECTA shield mounting medium (VECTOR Laboratories, #H-1000) Confocal microscope (OLYMPUS, FV1000) Cover glass (Matsunami, 24×50 mm, No. 1, 0.12 - 0.17)

[0095] 2. Preparation of reagents (Preparation of enzyme reaction solution) 0.4% Collagenase / HBSS and 2000 U / mL DNase / HBSS were mixed at a ratio of 1:1 to prepare an enzyme reaction solution (0.2% Collagenase / 1000 U / ml DNase / HBSS).

[0096] 3. Example 1 Example 1 is an example (AEPC administration group) for examining whether adipose-derived vascular endothelial (progenitor) cells exhibit a therapeutic effect in a stem cell depletion disease model animal.

[0097] 3-1. Cell preparation Adipose-derived vascular endothelial (progenitor) cells for animal experiments were prepared by the following steps (1) to (8). (1) Collection of fat, enzymatic treatment, and acquisition of SVF Subcutaneous fat was aseptically aspirated from a donor who had given informed consent. The obtained fat was allowed to stand or centrifuged to separate the fat layer, aqueous layer, and oil layer. The aqueous layer and oil layer were gently removed using a disposable pipette. The collected fat layer was dispensed into a conical tube, and the weight of the fat layer was weighed.

[0098] The enzyme reaction solution (0.2% Collagenase / 1000 U / mL DNase / HBSS) was pre-warmed at a temperature between room temperature and 37°C for 10 minutes. An equal volume of the enzyme reaction solution was added to the adipose layer obtained in (1) above, and the mixture was shaken at 37°C and 120 rpm for 30 minutes. The resulting enzyme reaction product was centrifuged at 800×g for 10 minutes. After centrifugation, the enzyme reaction product was separated into an oil layer, a residual adipose layer, an emulsion layer, an aqueous layer, and a cell pellet from the upper part of the centrifuge tube. The oil layer, adipose layer, emulsion layer, and aqueous layer were removed while taking care not to break the precipitated cell mass. The obtained cell pellet was gently suspended in 45 mL of HBSS (4°C) to obtain a cell suspension containing various adipose-derived cells.

[0099] A φ100 μm cell strainer was placed in a new 50 mL conical tube, and the above cell suspension (about 10 mL) together with the milky white fibrous mass was transferred onto the cell strainer, and the cell suspension was passed through the cell strainer. The fibrous mass trapped on the cell strainer was squeezed with the tip of a 5 mL disposable pipette to recover the cells attached to the fiber. The remaining cell suspension (about 35 mL) was passed through the cell strainer.

[0100] A φ40 μm cell strainer was placed in a new 50 mL conical tube, and the cell suspension passed through the 100 μmφ cell strainer was further passed through the φ40 μm cell strainer. The obtained cell suspension was centrifuged at 4°C and 800×g for 5 minutes. The supernatant was removed by suction, and the obtained cell pellet was resuspended in HBSS (45 mL) pre-cooled to 4°C. Further, the obtained cell suspension was centrifuged at 4°C and 800×g for 5 minutes. The supernatant was removed by suction. Through the above operations, the stromal vascular fraction (SVF) was obtained. Note that this stromal vascular fraction contains at least adipose-derived vascular endothelial (progenitor) cells, adipose-derived stem cells, and hematopoietic cells.

[0101] (2) Adherent culture of adipose-derived vascular endothelial (progenitor) cells (first time) The stromal vascular fraction (SVF) obtained in the above (1) was suspended in a medium (EGM-2 medium or EGM-2MV medium), and the number of nucleated cells and cell viability were counted using Luna-stem. 1×10 4 viable nucleated cells / 0.25 mL medium / cm 2 of cell density condition was seeded into a culture vessel and adherently cultured for 2 days under the conditions of 37 °C and 5% CO2. The medium was changed daily. By changing the medium, blood cell lineage cells, which are non-adherent cells, were removed. The adipose-derived vascular endothelial (precursor) cells contained in the adherent cells obtained by this adherent culture are referred to as "once adherently cultured adipose-derived vascular endothelial (precursor) cells" or "adipose-derived vascular endothelial (precursor) cells (P0) without passage culture".

[0102] Cell detachment was performed according to the following procedure. First, the adherent cells obtained by adherent culture were washed with HBSS (37 °C). Next, an appropriate amount of 1× TrypLE express (37 °C) (3 mL TrypLE express / 25 cm 2 ) was added and reacted at 37 °C and 5% CO2 for 5 minutes. Further, the reaction was stopped with a medium (EGM-2 medium or EGM-2MV medium), and a cell pellet (cell population) was obtained by centrifugation (4 °C, 300×g, 5 minutes). The number of nucleated cells was counted using Luna stem.

[0103] (3) Selection of CD31-positive cells by MACS (first time) The selection of cells presenting positive for CD31 was performed as follows according to the protocol of MACS of Miltenyi. The cell pellet obtained in the above (2) (1×10 7To a cell population containing less than cells, 60 μL of MACS buffer was added and gently resuspended. To the cell suspension, 20 μL of Fc receptor (FcR) block reagent was added and vortexed for 2 - 3 seconds. 20 μL of CD31 microbeads were added and gently pipetted, followed by a CD31 antibody reaction at 4°C for 15 minutes. 1 mL of MACS buffer was added and mixed by tapping, followed by centrifugation at 4°C and 300 × g for 3 minutes. The resulting cell pellet was resuspended in 0.5 mL of MACS buffer and temporarily stored at 4°C. A Miltenyi MS column was set on the separator and the column was washed once with 0.5 mL of MACS buffer. Immediately after column washing, the CD31 microbead-treated cell suspension was added to the column. 0.5 mL of MACS buffer was passed through the column three times. The flow-through was collected as a cell population containing CD31-negative cells (cells that are negative for CD31). Note that this cell population containing CD31-negative cells contains many adipose-derived stem cells. The fraction that was retained by the magnetic column and eluted by flushing after detaching the column from the separator was collected as a cell population (cell suspension) containing CD31-positive cells (cells that are positive for CD31). The resulting cell population containing CD31-positive cells was added to a new MS column, and the fraction that was retained by the magnetic column and eluted by flushing was collected as a cell population (cell suspension) containing CD31-positive cells. This cell population containing CD31-positive cells contains many adipose-derived vascular endothelial (progenitor) cells. The adipose-derived vascular endothelial (progenitor) cells contained in the CD31-positive cells obtained by this cell sorting are referred to as "once-sorted adipose-derived vascular endothelial (progenitor) cells".

[0104] (4) Adherent culture of adipose-derived vascular endothelial (progenitor) cells (second time) The cell population containing CD31-positive cells obtained in (3) above was resuspended in a medium (EGM-2 medium or EGM-2MV medium), and the number of nucleated cells and cell viability were counted using Luna-stem. 1 × 10 4 viable nucleated cells / 0.25 mL of medium / cm 2Seeded the culture vessel under the cell density condition of , and performed adherent culture for 3 days at 37 °C and 5% CO2. The medium was changed every other day. The adipose-derived vascular endothelial (precursor) cells contained in the adherent cells obtained by this adherent culture are referred to as "adipose-derived vascular endothelial (precursor) cells subjected to two adherent cultures" or "adipose-derived vascular endothelial (precursor) cells (P1) subjected to one passage culture". Cell detachment was performed in the same manner as in (2) described above.

[0105] (5) Selection of CD31-positive cells by MACS (second time) From the cell population obtained in (4) above, in the same manner as in (3) above, a cell population containing CD31-positive cells was selected by MACS. The adipose-derived vascular endothelial (precursor) cells contained in the CD31-positive cells obtained by this cell selection are referred to as "adipose-derived vascular endothelial (precursor) cells selected twice".

[0106] (6) Adherent culture of adipose-derived vascular endothelial (precursor) cells (third time) The cell population containing CD31-positive cells obtained in (5) above was resuspended in a medium (EGM-2 medium or EGM-2MV medium), and the number of nucleated cells and cell viability were counted with Luna-stem. 2.5×10 3 viable nucleated cells / 0.2 mL medium / cm 2 of the cell density condition, seeded in a culture vessel, and performed adherent culture at 37 °C and 5% CO2 until the confluence reached 80-90% (3-7 days). The medium was changed every two days. The adipose-derived vascular endothelial (precursor) cells contained in the adherent cells obtained by this adherent culture are referred to as "adipose-derived vascular endothelial (precursor) cells subjected to three adherent cultures" or "adipose-derived vascular endothelial (precursor) cells (P2) subjected to two passage cultures". Note that the obtained cell population contains a large amount and high purity of adipose-derived vascular endothelial (precursor) cells. Cell detachment was performed in the same manner as in (2) described above.

[0107] (7) Adherent culture of adipose-derived vascular endothelial (precursor) cells (fourth time) The cell population obtained in the above (6) was adherently cultured in the same manner as in the above (6). The adipose-derived vascular endothelial (precursor) cells contained in the adherent cells obtained by this adherent culture are referred to as "adipose-derived vascular endothelial (precursor) cells adherently cultured 4 times" or "adipose-derived vascular endothelial (precursor) cells subcultured 3 times (P3)". Cell detachment was carried out in the same manner as in the above (2).

[0108] (8) Adherent culture of adipose-derived vascular endothelial (precursor) cells (5th time) The cell population obtained in the above (7) was adherently cultured in the same manner as in the above (6). The adipose-derived vascular endothelial (precursor) cells contained in the adherent cells obtained by this adherent culture are referred to as "adipose-derived vascular endothelial (precursor) cells adherently cultured 5 times" or "adipose-derived vascular endothelial (precursor) cells subcultured 4 times (P4)". Cell detachment was carried out in the same manner as in the above (2).

[0109] For the obtained cell population, the positive rates of each surface antigen (CD45, CD34, CD31, CD146, CD105, and CD90) were measured using a flow cytometer. As a result, the positive rates of CD31, CD146, and CD105 were all 70% or more, and the positive rate of CD45 was less than 5%. In addition, the obtained cell population formed a tubular network structure in the network formation test. In addition, the obtained cell population contained cells that formed colonies in the colony formation test. From the above results, it was confirmed that the obtained cell population is a cell population containing adipose-derived vascular endothelial (precursor) cells. Note that the positive rates of CD34 and CD90 in the obtained cell population were both less than 5%. In addition, isolectin and von Willebrand factor were positive in the cell immunostaining test.

[0110] 3-2. Examination of the healing effect using a mouse model of a stem cell depletion disease In the experiments using the following three types of disease model mice, it was examined whether the "adipose-derived vascular endothelial (precursor) cells cultured by adherent culture five times" obtained in Example 1 showed a healing effect in the stem cell depletion disease model animals. Three types of disease model mice (A) Refractory skin ulcer in type 1 diabetes (B) Refractory skin ulcer in type 2 diabetes (C) Refractory skin ulcer due to radiation injury Regarding the therapeutic effect on "(B) Refractory skin ulcer in type 2 diabetes", in order to examine it in detail, it was carried out twice, so the two times will be described. (1) Preparation of a wound healing model using type 2 diabetes model mice Eight-week-old male type 2 diabetes mice (db / db mice, CLEA Japan, strain name: BKS.Cg-+Lepr db / + Lepr db / Jcl) were used, and a wound healing model (type 2 diabetes refractory ulcer model) was prepared according to the following procedure.

[0111] Nine-week-old db mice were anesthetized by isoflurane inhalation. The back of the anesthetized db mice was shaved with a clipper. Using a sterile biopsy punch (6.0 mm in diameter, Kai #BP-60F), the full thickness of the skin on the back of the db mice was peeled off to form a substantially circular wound area. To prevent wound contraction, a donut-shaped silicone splint (donut hole diameter: 9 mm) was placed on the wound area of the db mice and fixed using a size 6-0 nylon suture (Bare Medical, #BP11A06N-45).

[0112] (2) Administration The administration of adipose-derived vascular endothelial (precursor) cells to the wound healing model (type 2 diabetes refractory ulcer model) using type 2 diabetes model mice was carried out as follows. The "adipose-derived vascular endothelial (precursor) cells cultured by adherent culture five times" obtained in Example 1 were suspended using EGM-2MV medium, and cell suspension a1 containing 1.0×10 6 adipose-derived vascular endothelial (precursor) cells / mL, and 4.0×10 6Cell suspensions a2 containing adipose-derived vascular endothelial (precursor) cells at [X] cells / mL were each prepared. Next, the above cell suspension a1 or a2 and 0.4% sodium hyaluronate (Wako Pure Chemical Industries, #089-10343) were mixed at a ratio of 1:1, and [X] 5 cells / mL of adipose-derived vascular endothelial (precursor) cells to obtain administration solution A1, and [X] 6 cells / mL of adipose-derived vascular endothelial (precursor) cells to obtain administration solution A2. Administration solutions A1 and A2 with a dosage of 0.2 mL / mouse were administered subcutaneously at 4 to 8 locations around the wound of the wound healing model prepared in (1) above. That is, the number of administered cells in the experimental system using administration solution A1 was [X] 5 cells / mouse for adipose-derived vascular endothelial (precursor) cells, and the number of administered cells in the experimental system using administration solution A2 was [X] 5 cells / mouse for adipose-derived vascular endothelial (precursor) cells.

[0113] 4. Example 2 Example 2 is an example (AEPC + ASC combined administration group) for examining whether the healing effect is enhanced in a stem cell depletion disease model animal when adipose-derived vascular endothelial (precursor) cells are used together with adipose-derived stem cells. 4-1. Cell preparation Adipose-derived stem cells for use in animal experiments together with adipose-derived vascular endothelial (precursor) cells were prepared by the following steps (1) to (4).

[0114] (1) Adipose collection, enzymatic treatment, and acquisition of SVF SVF was obtained in the same manner as in “(1) Adipose collection, enzymatic treatment, and acquisition of SVF” in Example 1.

[0115] (2) Adherent culture of adipose-derived stem cells (first time) The stromal vascular fraction (SVF) obtained in (1) above was suspended in DMEM / F12 medium (medium for adipose-derived stem cells) containing 10% FBS and Penicillin-Streptomycin, and the number of nucleated cells and cell viability were counted using Luna-stem. 4000 cells / cm 2 Please note that the specific cell numbers in the original text are represented by placeholders (e.g., [X]) and need to be filled with the actual cell numbers in the translation. Also, the 7-digit tags ( 5 , 6 , etc.) are preserved as they are.Seeded in a culture vessel under the cell density condition, and subjected to adherent culture at 37°C and 5% CO2 until the confluency reached 80 - 90%. Note that one or two days after seeding the stromal vascular fraction (SVF), it was washed three times with HBSS or DPBS pre-warmed to 37°C to remove hematopoietic cells, which are non-adherent cells. In addition, the medium was changed every three days. By changing the medium, hematopoietic cells, which are non-adherent cells, were further removed. The adipose-derived stem cells contained in the adherent cells obtained by this adherent culture are referred to as "adipose-derived stem cells subjected to one adherent culture" or "adipose-derived stem cells at passage 0 (P0)".

[0116] Cell detachment was performed according to the following procedure. First, the adherent cells obtained by adherent culture were washed with HBSS (37°C). Next, an appropriate amount (3 mL TrypLE express / 25 cm 2 ) of 1× TrypLE express (37°C) was added, and the reaction was carried out at 37°C and 5% CO2 for 5 minutes. Furthermore, the reaction was stopped with the medium for adipose-derived stem cells, and a cell pellet (cell population) was obtained by centrifugation (4°C, 300×g, 5 minutes). The number of nucleated cells was counted using Luna stem.

[0117] (3) Adherent culture of adipose-derived stem cells (second time) The cell population obtained in (2) above was subjected to adherent culture in the medium for adipose-derived stem cells in the same manner as in (2) above. The adipose-derived stem cells contained in the adherent cells obtained by this adherent culture are referred to as "adipose-derived stem cells subjected to two adherent cultures" or "adipose-derived stem cells at passage 1 (P1)". Cell detachment was performed in the same manner as in (2) above.

[0118] (4) Adherent culture of adipose-derived stem cells (third time) The cell population obtained in (3) above was subjected to adherent culture in the medium for adipose-derived stem cells in the same manner as in (2) above. The adipose-derived stem cells contained in the adherent cells obtained by this adherent culture are referred to as "adipose-derived stem cells subjected to three adherent cultures" or "adipose-derived stem cells at passage 2 (P2)". Cell detachment was performed in the same manner as in (2) above.

[0119] For the obtained cell population, the positive rates of each surface antigen (CD90, CD73, CD105, CD31, and CD45) were measured using a flow cytometer. As a result, the positive rates of CD90, CD73, and CD105 were all 80% or more, and the positive rates of CD31 and CD45 were both less than 5%. From the above results, it was confirmed that the obtained cell population was a cell population containing adipose-derived stem cells.

[0120] 4-2. Examination of the healing effect using a stem cell depletion disease model mouse In the experiment shown below, the "adipose-derived vascular endothelial (progenitor) cells cultured by adhesion 5 times" obtained in Example 1 and the "adipose-derived stem cells cultured by adhesion 3 times" obtained in Example 2 were administered to a stem cell depletion disease model mouse to examine whether they showed a healing effect.

[0121] (A) Wound healing effect on type 1 diabetic intractable skin ulcers (1) Induction of type 1 diabetes Five-week-old male immunodeficient SCID mice (CLEA Japan, strain name: C.B-17 / Icr-scid / scidJcl) and their control wild-type mice (CLEA Japan, strain name: C.B17 / Icr-+ / +Jcl) were acclimated and then weighed. During the period until the end of the experiment, the body weight was measured weekly to evaluate the overall condition of the mice, and the progress was recorded. All the tests were performed under isoflurane (Fizer) inhalation anesthesia. After 24-hour fasting, a small amount (1 μL) of fresh blood was collected from the tail vein for glucose concentration measurement, and the blood glucose concentration was measured. The blood glucose level was measured using equipment (Terumo, Medisure Fit Pro II, #MS-FKP02, and Iwai Chemicals, Glucose Pilot, #GP-01). Streptozotocin (STZ; Sigma Aldrich catalog #S0130) was dissolved in citrate buffer (pH = 4.5) and sterilized with a 0.22 μm syringe filter to prepare a sterilized STZ solution. The sterilized STZ solution was administered intraperitoneally (dose: 150 mg / kg) to induce type 1 diabetes. Also, as a negative control, a group that received intraperitoneal administration of sterilized citrate buffer was prepared. Twenty-four hours after the first administration of STZ, the sterilized STZ solution was administered intraperitoneally again (dose: 150 mg / kg). The negative control group received intraperitoneal administration of sterilized citrate buffer again 24 hours after the first administration of sterilized citrate buffer. Three days after the administration of STZ, the blood glucose concentration was measured. When the blood glucose concentration was 300 mg / dl or higher, the preparation of the type 1 diabetic mouse model was completed. When the blood glucose concentration was below 300 mg / dl, an additional 150 mg / kg of STZ was administered intraperitoneally, and the onset of diabetes was measured with a blood glucose measuring device 3 days later.

[0122] (2) Preparation of a wound healing model using type 1 diabetic model mice A wound healing model was established in 9-week-old type 1 diabetes-induced immunodeficient SCID mice with a blood glucose concentration of 300 mg / dl or higher. All wound healing tests were performed under isoflurane inhalation anesthesia. The back was shaved with a clipper. For the purpose of preventing wound contraction, a donut-shaped silicon splint (Kyowa Kogyo, inner diameter of donut hole 9 mm, outer diameter 15 mm, thickness 1 mm) was fixed with a size 6-0 nylon suture (Bare Medical, #BP11A06N-45). The entire skin layer was peeled off in a substantially circular shape using a sterile biopsy punch (Kai biopsy trephine 6 mm #BP-60F, 6.0 mm diameter).

[0123] (3) Administration Administration of adipose-derived vascular endothelial (progenitor) cells to a type 1 diabetes intractable ulcer model using type 1 diabetes SCID mice was performed as follows. The "adipose-derived vascular endothelial (progenitor) cells cultured by adhesion 5 times" obtained in Example 1 were suspended using EGM-2MV medium to prepare cell suspension a1 containing 1.0×10 6 adipose-derived vascular endothelial (progenitor) cells per mL, and cell suspension a2 containing 4.0×10 6 adipose-derived vascular endothelial (progenitor) cells per mL, respectively. Next, the above cell suspension a1 or a2 and sterile 0.4% (v / w) sodium hyaluronate (Wako Pure Chemical Industries, #089-10343) dissolved in complete medium for microvascular endothelial cells (Lonza, EGM-2MV, #CC-3202) were mixed at a ratio of 1:1 to prepare administration solution A1 containing 5.0×10 5 adipose-derived vascular endothelial (progenitor) cells per mL, and administration solution A2 containing 2.0×10 6 adipose-derived vascular endothelial (progenitor) cells per mL. Administration solutions A1 and A2 with a dosage of 0.2 mL / mouse were injected subcutaneously at 4 to 8 locations around the wound of the wound healing model prepared in (1) above. That is, the number of administered cells in the experimental system using administration solution A1 was 1.0×10 5 adipose-derived vascular endothelial (progenitor) cells per mouse, and the number of administered cells in the experimental system using administration solution A2 was 4.0×10 5It was per mouse. The animal experiment started with 5 animals in each group, both the experimental group and the control group. Until the cell administration test on mice was completed, the human cell - 0.2% (w / v) sodium hyaluronate - EGM - 2MV medium solution was stored in ice - cold condition, and after administration, the viability of the administered cells was confirmed. Using a cell counter (Logos biosystems, LUNA - STEM), the cell viability (%) was measured with Propidium iodide / Acridine orange.

[0124] Comparative Example 1 Comparative Example 1 is a control experiment (Vehicle administration group) in which a cell - free administration solution (Vehicle) was administered to a stem - cell - depleted disease model animal.

[0125] Preparation of Vehicle EGM - 2MV medium and 0.4% (w / v) sodium hyaluronate - EGM - 2MV medium were mixed at a ratio of 1:1 to prepare a 0.2% (w / v) sodium hyaluronate - EGM - 2MV medium solution, which is a cell - free administration solution (Vehicle).

[0126] (4) Observation of the wound - healing effect over time and image analysis Taking the administration day as day 0, the progress of the wound area on days 0, 3, 7, 10, 14, 17, and 21 was macro - photographed with a single - lens reflex camera. After photographing, a dressing agent was used to prevent the silicon splint from falling off, the wound from drying, and scab formation. The macro - photos were analyzed with Adobe Photoshop 2020 (Adobe creative cloud). After converting the resolution to 300 pixel / inch, the measurement scale was set such that 1 pixel = 1 pixel. The inner area of the donut - shaped splint and the wound area were selected, and the number of pixels of the selected area was measured using a measurement tool. The wound - healing area ratio was calculated by the following formula. Wound healing(%) = (Area of the wound in pixels on day X÷Area of the inside of the splint in pixels)÷(Area of the wound in pixels on day 0÷Area of the inside of the splint in pixels)×100

[0127] Using Excel software (Microsoft, Excel for mac 2016), the mean value and standard deviation value (SD) of each group were calculated from the values of the wound healing area ratio of each mouse. For the significance test, statistical analysis software (Graphpad, Prism 6 for MAC OS X, version 6.0d) was used to perform One-way ANOVA with Tukey’s test.

[0128] (5) Collection of histological specimens from the wound healing site After taking macrophotographs on the 21st day after administration, the entire skin layer was peeled off in a roughly circular shape with a biopsy punch (Kai biopsy trephine 8.0 mm, #BP-80F) with a diameter of 8 mm inside a donut-shaped silicon splint (diameter 9 mm) and collected. The collected skin pieces included the skin at the wound healing site and the normal skin around it. The skin pieces were fixed with a fixative (BD Pharmingen IHC Zinc Fixative #550523) for 24 hours at 4°C. The roughly circular skin pieces were cut with a scalpel through the center and embedded in paraffin in the direction where the cut surface of the skin by the scalpel was flat. Also, human skin tissue used as a control was fixed and embedded in paraffin in the same procedure.

[0129] (6) Confirmation of engraftment of administered human cells Whether the administered AEPC and ASC engrafted into the type 1 diabetic SCID mice was confirmed by immunostaining using a human-specific Golgi antibody. As the human-specific Golgi antibody, Anti-TGOLN2 antibody produced in rabbit (Sigma-Aldrich, #HPA012723) was selected. Trans-Golgi Network Protein 2 (TGOLN2) is one of the proteins expressed in the human Golgi apparatus. The peptide sequence used as the antigen for this antibody is publicly available from the vendor Sigma-Aldrich. When the antigen sequence was searched using the browser software Standard Protein BLAST (NIH National Institutes of Health, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins), the animal species with complementarity and detected were limited to primates and monkeys such as chimpanzee (Pan troglodytes), bonobo (Pan paniscus), and western lowland gorilla (Gorilla gorilla gorilla), and it was confirmed that there was no complementarity with the protein sequence expressed in mice. Therefore, it can be said that it is an antibody that specifically detects the Golgi apparatus of human cells, and it was selected considering that it can detect the presence or absence of engraftment of human cells administered to mouse tissues.

[0130] Procedure for immunostaining The specimen paraffin block was sectioned into 4-μm thick slices using a microtome, and then underwent deparaffinization and rehydration treatments. Antigen retrieval (VECTOR, Antigen unmasking solution #H-3300) was performed according to the kit protocol. After washing with PBS (Wako, FUJIFILM, #162-18547), permeabilization was carried out with 0.025% (w / v) Triton-X100 / PBS for 5 minutes at room temperature. After washing with PBS, endogenous peroxidase was blocked with 3% (v / v) H2O2 / H2O for 30 minutes at room temperature. After washing with PBS, blocking was performed with 2.5% Normal horse serum included in the immunostaining kit (VECTOR, ImmPRESS® Horse Anti-Rabbit IgG Polymer Kit #MP-7401-50) for 20 minutes at room temperature. Further blocking was carried out with 5% (w / v) skim milk (Nacalai Tesque, #31149-75) / PBS for 15 minutes at room temperature. The antibody reaction was carried out by preparing the human Golgi body antibody (Sigma-Aldrich, Anti-TGOLN2 antibody produced in rabbit, #HPA012723) at a concentration of 0.8 μg / mL with an antibody diluent (Agilent, Dako antibody diluent #S0809-83) and reacting it at 4°C for 16 hours. As one of the negative controls, normal IgG from the same species (Thermo, Rabbit IgG Isotype Control, #02-6102) was prepared at a concentration of 0.8 μg / mL with the antibody diluent and reacted at 4°C for 16 hours.

[0131] Color development was performed using the peroxidase / DAB reaction (VECTOR, ImmPACT(R) DAB #SK-4105). Methyl green nuclear staining (VECTOR, #H-3402) was performed as a counterstain. Dehydration, clearing, and mounting were carried out. Imaging was performed using an inverted microscope (Keyence, All-in-One microscope, #BZX-710, lens x20 PlanApoλ NA0.75) with the exposure fixed at 1 / 400 seconds for bright-field photography. For the field of view of the immunostained image, a boundary position including a part of the scar and normal skin was selected with the healed scar as the center.

[0132] The immunostaining conditions are shown in Table 1 below. a - i correspond to the stained images of each condition in Figure 3.

[0133]

Table 1

[0134] <Results> The cell viability of the human cell - 0.2% (w / v) sodium hyaluronate - EGM - 2MV medium solution after the cell administration test to mice was 94% or more under all administration conditions. The animal experiment was carried out as shown in Table 2 below. On the 0th day of the test (D0), 5 mice were prepared for each group and the test was started. On the 21st day of the test end (D21), some mice died under some conditions, and each group had 4 - 5 mice. For the measurement results of the wound healing area rate in the wound healing test and the immunostaining results for confirming the engraftment of the administered human cells in mice, the dead mice were not included, so the results were obtained with n = 4 - 5 as shown in Table 2.

[0135]

Table 2

[0136] Figure 1 shows the course of wound healing in a type 1 diabetic refractory ulcer model when low - volume (1.0×10 5 cells / mouse) adipose - derived vascular endothelial (precursor) cells were administered. As shown in Fig. 1, regarding the wound healing area ratio on Day 7 and Day 10, the adipose-derived vascular endothelial (progenitor) cell (AEPC) alone group and the AEPC + adipose-derived stem cell (ASC) combined administration group showed a tendency for improvement compared to the Vehicle administration group. Furthermore, regarding the wound healing area ratio on Day 14, Day 17, and Day 21, the AEPC alone group showed a tendency for improvement compared to the Vehicle administration group, and the AEPC + ASC combined administration group showed a significant improvement. Also, regarding the wound healing area ratio on Day 14 and Day 17, the AEPC + ASC combined administration group showed a tendency for improvement superior to that of the non-induced type 1 diabetes (normal) SCID mouse group (Fig. 1-1. SCID / citrate buffer-None group. A group that heals wounds without developing intractable ulcers caused by type 1 diabetes).

[0137] Fig. 2 shows the course of wound healing in a type 1 diabetic intractable ulcer model when high-volume (4.0×10 5 cells / mouse) adipose-derived vascular endothelial (progenitor) cells were administered.

[0138] As shown in Fig. 2, regarding the wound healing area ratio on Day 7 and Day 10, the AEPC administration group and the AEPC + ASC combined administration group showed a tendency for improvement compared to the Vehicle administration group. Furthermore, regarding the wound healing area ratio on Day 14, Day 17, and Day 21, the AEPC administration group and the AEPC + ASC combined administration group showed a significant improvement compared to the Vehicle administration group. Also, regarding the wound healing area ratio on Day 14 and Day 17, the AEPC administration group and the AEPC + ASC combined administration group showed a tendency for improvement superior to that of the non-induced type 1 diabetes (normal) SCID mouse group (Fig. 1-1. SCID / citrate buffer-None group. A group that heals wounds without developing intractable ulcers caused by type 1 diabetes). Therefore, it was clarified that the cell population containing AEPC (AEPC administration group) shows a healing effect in a type 1 diabetic intractable ulcer model. Furthermore, it was clarified that the cell population containing AEPC and ASC (AEPC + ASC combined administration group) shows an even higher healing effect. Also, for low-volume (1.0×10 5Even in the AEPC group (number / mouse), it was suggested that a sufficient healing effect was exhibited. Regarding the wound healing area ratio, the ASC administration group did not show a tendency for improvement compared to the Vehicle administration group. In the literature so far, regarding the wound healing area ratio, it has been reported many times that the ASC single group has a higher therapeutic effect compared to the Vehicle administration group. However, the composition of the Vehicle used in the Vehicle administration group in this study was a solution of sodium hyaluronate in a complete medium for microvascular endothelial cells (Lonza, EGM-2MV), and multiple growth factors (human fibroblast growth factor basic, epidermal growth factor, insulin -like growth factor, Ascorbic acid, hydrocortisone, fetal bovine serum) that accelerate tissue remodeling contained in EGM-2MV were released in a sustained-release form. Since the Vehicle administration group itself was already a condition that accelerated wound healing, it was suggested that the effect of the ASC single group was difficult to observe. In other words, it was suggested that the AEPC single group and the AEPC+ASC combined administration group had a higher therapeutic effect than the Vehicle administration group and the ASC single group in this study, which were conditions that accelerated wound healing.

[0139] The results regarding the engraftment of human cells into type 1 diabetic SCID mice were shown in Table 3 and Figure 3 by immunostaining using a human-specific Golgi antibody.

[0140]

Table 3

[0141] The bars in Figure 3 indicate 100 μm. The arrow indicates a representative example of human-specific Golgi antibody-positive cells. The immunostained images were taken by selecting a boundary position that includes a part of the scar and normal skin area centered on the healed scar, that is, the engraftment of human cells in the wound healing scar area is discussed here. Figures 3a and 3b are the negative control groups reacted with normal rabbit IgG, and the results showed negative, indicating no non-specific reaction of immunostaining. Figure 3c is the positive control group reacted with human-specific TGOLN2 antibody to human skin tissue, and the results showed positive, indicating the detection of TGOLN2 of human cells. Since Figure 3d is the Vehicle group without administration of human cells, the degree of non-specific immunoreaction of human-specific TGOLN2 antibody to mouse tissue was verified, and a lighter and less distinct DAB staining color development than the positive cells indicated by the arrow in Figure 3d was confirmed. From Figures 3a, b, c, and d above, it can be said that the present test system verified the presence or absence of cell engraftment using a tissue immunostaining method that detects TGOLN2 specifically expressed in human Golgi apparatus and has low non-specific detection in mouse tissue. The faint non-specific color development of DAB indicated by the arrow in Figure 3d was used as the background. In the ASC single administration group of Figure 3e, it was shown that ASC-derived cells were engrafted in the dermis and subcutaneous adipose tissue. In the AEPC single administration groups of Figures 3f and 3g, it was shown that AEPC-derived cells were engrafted mainly with a lumen structure in the dermis and subcutaneous adipose tissue. In the combined administration group of AEPC and ASC in Figures 3h and 3i, it was confirmed that AEPC-derived cells and ASC-derived cells were engrafted in the dermis and subcutaneous adipose tissue. From the above, when a full-thickness skin defect was created in immunodeficient SCID mice induced with type 1 diabetes and human cells were transplanted subcutaneously, it was suggested that the transplanted human ASC-derived cells and human AEPC-derived cells were engrafted as verified 21 days later.

[0142] (B) Wound healing effect on intractable skin ulcers in type 2 diabetes (1) Preparation of a wound healing model using type 2 diabetic model mice 8-week-old male type 2 diabetic mice (db / db mice, CLEA Japan, strain name: BKS.Cg-+Lepr db / + Lepr db / Jcl), and its heterozygous mice (+ / db mice, CLEA Japan, strain name: BKS.Cg-m + / + Lepr db After acclimatizing the db and its heterozygous mice (+ / db mice, CLEA Japan, strain name: BKS.Cg-m + / + Lepr / Jcl), body weight measurements were taken. During the period until the end of the experiment, body weight measurements were taken weekly to evaluate the overall condition of the mice, and the progress was recorded. After 24 hours of fasting, a small amount (1 μL) of fresh blood was collected from the tail vein for glucose concentration measurement, and the blood glucose concentration was measured using an instrument (Iwai Chemicals, Glucose Pilot, #GP-01). It was confirmed that the blood glucose concentration of the type 2 diabetic mice was 300 mg / dL or higher (diabetes), and that of the heterozygous mice was 200 mg / dL or lower (normal). A wound healing model was created at 9 weeks of age. All mouse experiments were performed under isoflurane inhalation anesthesia. The back was shaved with a clipper. For the purpose of preventing wound contraction, a donut-shaped silicon splint (Kyowa Industry, inner diameter of donut hole 9 mm, outer diameter 15 mm, thickness 1 mm) was fixed with a size 6-0 nylon suture (Bare Medical, #BP11A06N-45). The entire skin layer was peeled off in a roughly circular shape using a sterilized biopsy punch (Kai Biopsy Trephine 6 mm #BP-60F, 6.0 mm diameter).

[0143] The methods of "administration" to the type 2 diabetic refractory ulcer model, "time-course observation and image analysis of wound healing effect", "collection of histological specimens from the wound healing site", and "confirmation of engraftment of administered human cells" were carried out in the same manner as for the type 1 diabetic refractory ulcer model. The results of this test were listed because the test was conducted twice.

[0144] <Results> The cell viability of the human cell - 0.2% (w / v) sodium hyaluronate - EGM-2MV medium solution after the cell administration test to mice was 94% or higher under all administration conditions. The first test was conducted as shown in Table 4 below. On the 0th day of the test (D0), 5 mice were prepared for each group and the test was started. Until the 21st day of the test end day (D21), there were no deaths, etc., and each group obtained the measurement results of the wound healing area ratio in the wound healing test with 5 mice.

[0145]

Table 4

[0146] Figure 4 shows the course of wound healing in a type 2 diabetic intractable ulcer model when low-dose (1.0×10 5 cells / mouse) of adipose-derived vascular endothelial (precursor) cells were administered. Figure 4 As shown in Figure 4 , for the wound healing area ratio on Day 10 and Day 14, the AEPC administration group and the AEPC+ASC combined administration group showed a tendency to improve compared with the Vehicle administration group. Furthermore, for the wound healing area ratio on Day 17, the AEPC administration group showed a tendency to improve compared with the Vehicle administration group, and the AEPC+ASC combined administration group showed a significant improvement.

[0147] Figure 5 shows the course of wound healing in a type 2 diabetic intractable ulcer model when high-dose (4.0×10 5 cells / mouse) of adipose-derived vascular endothelial (precursor) cells were administered. Figure 5 As shown in Figure 5 , for the wound healing area ratio on Day 10, the AEPC+ASC combined administration group showed a tendency to improve compared with the Vehicle administration group. Also, for the wound healing area ratio on Day 14, the AEPC administration group showed a tendency to improve compared with the Vehicle administration group, and the AEPC+ASC combined administration group showed a significant improvement. Furthermore, for the wound healing area ratio on Day 17, the AEPC administration group showed a tendency to improve compared with the Vehicle administration group, and the AEPC+ASC combined administration group showed a significant improvement.

[0148] As shown in Table 5 below Conduct the second test Five mice per group were prepared and the test was started on the 0th day of the test (D0). Until the 21st day (D21) of the end of the test, there were no deaths, etc. Each group obtained the measurement results of the wound healing area ratio of the wound healing test with 5 mice, and the immunostaining results for confirming the engraftment of the administered human cells into the mice.

[0149]

Table 5

[0150] Figure 6 shows the course of wound healing in a type 2 diabetic intractable ulcer model when low-dose (1.0×10 5 cells / mouse) adipose-derived vascular endothelial (precursor) cells were administered. As shown in Figure 6, regarding the wound healing area ratio on Day 7 and Day 10, the AEPC+ASC combined administration group showed a significant improvement compared to the Vehicle administration group. Figure 7 shows the course of wound healing in a type 2 diabetic intractable ulcer model when high-dose (4.0×10 5 cells / mouse) adipose-derived vascular endothelial (precursor) cells were administered. As shown in Figure 7, regarding the wound healing area ratio on Day 10, Day 14, and Day 17, the AEPC+ASC combined administration group showed a tendency of improvement compared to the Vehicle administration group.

[0151] The results regarding the presence or absence of engraftment of human cells into type 2 diabetic mice were shown in Table 6 and Figure 8 by immunostaining using a human-specific Golgi antibody. In Figure 8, Bars indicate 100 μm. Arrows indicate representative examples of human-specific Golgi antibody-positive cells.

[0152]

Table 6

[0153] The immunostained images were taken by selecting a boundary position that includes a part of the scar and normal skin centered on the healed scar, that is, the engraftment of human cells in the wound healing scar area is discussed here. Figures 8a and 8b are the negative control groups reacted with normal rabbit IgG, and the results showed negative, indicating no non-specific reaction of immunostaining. Figure 8c is the positive control group reacted with a human-specific TGOLN2 antibody to human skin tissue, and the results showed positive, indicating the detection of TGOLN2 in human cells. Figure 8d is the Vehicle group without administration of human cells, so the degree of non-specific immunoreaction of the human-specific TGOLN2 antibody to mouse tissue was verified, and the results showed a less clear and lighter DAB staining color development than that of positive cells. From Figures 8a, b, c, and d above, it can be said that this test system uses a tissue immunostaining method that can detect TGOLN2 specifically expressed in the human Golgi apparatus and has low non-specific detection in mouse tissue to verify the presence or absence of human cell engraftment. The faint non-specific color development of DAB seen in Figure 8d was used as the background. In the ASC-alone administration group of Figure 8e and the high-dose AEPC-alone administration group of Figure 8f, it was shown that ASC-derived cells and AEPC-derived cells had engrafted in the dermis and subcutaneous adipose tissue. In the low-dose AEPC-alone administration group of Figure 8f, it was shown that AEPC-derived cells had engrafted mainly with a lumen structure in the dermis and subcutaneous adipose tissue. In the combined administration groups of AEPC and ASC in Figures 8h and 8i, it was confirmed that AEPC-derived cells and ASC-derived cells had engrafted in the dermis and subcutaneous adipose tissue. However, the number of engrafted cells was less under all administration conditions than when human cells were administered to type 1 diabetic SCID mice. As a reason for this, although there are differences between type 1 and type 2 diabetes diseases, it was considered that immunity was involved. In the type 1 diabetes model, it is a test system in which human cells are administered to immunodeficient SCID mice lacking T cells and B cells, and it is difficult for human cells to be excluded by the immune reaction. In contrast, in the type 2 diabetes model, db / db mice with a normal immune reaction are used, so the administered human cells are excluded by the immune reaction, and only the human cells that could not be completely excluded by the immune reaction engrafted and were detected by immunostaining, suggesting that the number of engrafted cells was small.Even in the wound healing test, in the type 1 diabetes model, the wound healing area ratio was significantly improved in the human cell administration group compared to the Vehicle group, while in the type 2 diabetes model, the wound healing area ratio only showed a tendency to improve in the human cell administration group compared to the Vehicle group. From the above, it was revealed that a cell population containing adipose-derived vascular endothelial (progenitor) cells and adipose-derived stem cells showed a tendency to improve the healing effect on intractable skin ulcers derived from type 2 diabetes.

[0154] (C) Wound healing effect on radiation-induced intractable skin ulcers (1) Preparation of radiation-induced nude mice Seven-week-old male nude mice (Japan CLEA, strain name: BALB / cAJcl-nu / nu) were acclimated for 24 hours and then locally irradiated with radiation under isoflurane inhalation anesthesia. The whole body of the lying mice was shielded with a lead plate molded into a dome shape, the back skin was pulled out from the lead plate shielding, and the skin at the pulled-out site was irradiated once a week at 5 Gy for 8 weeks. The skin was pulled out from the same location each time and irradiated with radiation. The non-irradiated group was only anesthetized. During the period until the end of the experiment, the body weight was measured weekly to evaluate the overall condition of the mice, and the progress was recorded.

[0155] (2) Preparation of a wound healing model using radiation-induced model mice The wound healing test was carried out 12 weeks after the end of irradiation. The method was the same as that of "Preparation of a wound healing model using a type 1 diabetes model". The methods of "administration" to the radiation-induced intractable ulcer model and "time-course observation and image analysis of the wound healing effect" were carried out in the same way as those for the type 1 diabetes intractable ulcer model. The cell administration conditions are shown in Table 7 below.

[0156]

Table 7

[0157] <Results> Figure 9 shows the process of wound healing in a radiation-induced intractable skin ulcer model. As shown in Figure 9, regarding the wound healing area ratio on Day 7, it was significantly improved in the AEPC alone group (E2) and the AEPC + ASC combined administration group (E6) compared to the Vehicle administration group (C2). Regarding the wound healing area ratio on Day 7, there was a tendency for improvement in the AEPC alone groups (E3, E4) and the AEPC + ASC combined administration group (E5) compared to the Vehicle administration group (C2). Regarding the wound healing area ratio on Day 11, it was significantly improved in the ASC alone administration group (E1), the AEPC alone groups (E3, E4), and the AEPC + ASC combined administration groups (E5, E6) compared to the Vehicle administration group (C2). Regarding the wound healing area ratio on Day 14, it was significantly improved in all experimental groups compared to the Vehicle administration group. In the AEPC + ASC combined administration groups (E5, E6), the wound area was significantly smaller compared to the groups administered alone.

[0158] From the above, it was clarified that the cell population containing adipose-derived vascular endothelial (progenitor) cells shows a wound healing effect on intractable skin ulcers derived from radiation injury. It was clarified that the cell population containing adipose-derived vascular endothelial (progenitor) cells and adipose-derived stem cells shows an even higher healing effect.

[0159] <Conclusion> It was clarified that the cell population containing adipose-derived vascular endothelial (progenitor) cells (AEPC) shows a wound healing effect on intractable skin ulcer models of type 1 diabetic SCID mice, type 2 diabetic db / db mice, and radiation-induced nude mice. It was clarified that the cell population containing adipose-derived vascular endothelial (progenitor) cells and adipose-derived stem cells (AEPC + ASC) shows an even higher healing effect.

[0160] <Manufacture of Pharmaceutical Composition> A part of the cell populations of Examples 1 and 2 is used for the preparation of a pharmaceutical composition. 4×10 6 adipose-derived vascular endothelial (progenitor) cells, 1×10 6Prepare a pharmaceutical composition containing individual adipose-derived stem cells and 2 mL of a 0.2% sodium hyaluronate-EGM-2MV medium solution. The above pharmaceutical composition can be administered subcutaneously to a patient or subject.

Claims

**Claim 1** A pharmaceutical composition for preventing and / or treating a stem cell-depleting disease, comprising a cell population containing cultured adipose-derived vascular endothelial (precursor) cells and a pharmaceutically acceptable medium, wherein the stem cell-depleting disease is type 1 diabetic ulcer, type 2 diabetic ulcer, refractory ulcer, skin ulcer or radiation ulcer, and the cell population contains 50% or more of the adipose-derived vascular endothelial (precursor) cells. **Claim 2** The pharmaceutical composition according to claim 1, wherein the adipose-derived vascular endothelial (precursor) cells are adipose-derived vascular endothelial precursor cells that have been adherently cultured at least twice. **Claim 3** The pharmaceutical composition according to claim 1 or 2, wherein the cell population further contains cultured adipose-derived stem cells. **Claim 4** The pharmaceutical composition according to claim 3, wherein the cell population contains 12.5% or more and 50% or less of the adipose-derived stem cells.

Images